Generation of a lineage specific cell from a primate extended blastocyst cell line

ABSTRACT

An isolated primate embryonic cell is provided as well as cell cultures and cell lines derived therefrom. Also provided are methods of generating and using such cells.

RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.11/665,248 filed on Apr. 12, 2007, which is a National Phase of PCTPatent Application No. PCT/IL2005/001074 having International FilingDate of Oct. 11, 2005, which claims the benefit of priority of U.S.Provisional Patent Application No. 60/617,045 filed on Oct. 12, 2004.The contents of the above applications are all incorporated herein byreference.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to cells derived from delayed blastocystsand to cell lines generated therefrom.

Embryonic development starts soon after fertilization with blastomercleavage, proliferation and differentiation. The blastomers within thedeveloping mammalian embryo remain totipotent until the morulacompaction stage. In the compacted embryo, the blastomers initiatepolarization which results in two distinct cell-populations; the innercell mass (ICM), which contributes to the embryo, and the outertrophectoderm layer, which develops into the extra embryonic layers. Itis at this stage of embryogenesis—towards the end of the first week ofdevelopment that embryonic stem (ES) cells are traditionally derivedfrom the inner cell mass of the blastocyst.

At the time of implantation, the ICM is separated into a layer ofprimitive endoderm, which gives rise to the extra embryonic endoderm,and a layer of primitive ectoderm, which gives rise to the embryo properand to some extra embryonic derivatives [Gardner J. EmbryologicalExperiment and Morphology, 1982; 68: 175-198]. During the next majorphase of development, termed gastrulation, the embryonic ectodermdifferentiates into the three primary germ layers—endoderm (insidelayer), mesoderm (middle layer), and ectoderm (outer layer). The cellsbecome progressively restricted to a specific lineage, losing theirpluripotency and thus are regarded as multi-potent progenitor cells.Therefore, pluripotent embryonic stem cells proliferate and replicate inthe intact embryo only for a limited period of time.

Embryonic stem cells are characterized by their ability to propagateindefinitely in culture, as undifferentiated cells, while they can beinduced to differentiate in vivo into teratomas when injected into SClDmice [Thomson, J. A., et al., (1998), Science, 282, 1145-1147;Reubinoff, B. E., et al., (2000), Nature Biotechnol., 18, 399-404]. Theymay also differentiate in vitro into embryoid bodies (EBs) that containembryonic cells from the three germ layers (endoderm, mesoderm, andectoderm). Moreover, this differentiation can be somewhat directed bythe addition of growth factors into the culture media.

Human ES cells may also be genetically manipulated in culture [Eiges etal., (2001) Curr. Biol., 11, 514-518] and the transfected cells remainpluripotent and retain a normal karyotype [Shuldiner et al., (2003) StemCells 21, 257-265].

As a result of their unique features, it has been suggested that humanES cells hold the promise of changing the face of cell transplantation,by replacing or restoring tissue that has been damaged by disease orinjury. Replacement of non-functional cells using ES cells technologycan offer a lifelong treatment. Thus, diseases that might be treated bytransplanting human ES-derived cells include Parkinson's disease,diabetes, traumatic spinal cord injury, Purkinje cell degeneration,Duchenne's muscular dystrophy, heart failure, and osteogenesisimperfecta.

Many other potential uses of human ES cells have been proposed that donot involve transplantation. For example, human ES cells could be usedto study early events in human development. Human ES cells could also beused to test candidate therapeutic drugs or potential toxins bydirecting their differentiation into specific cell types. ES-derivedcells may be more likely to mimic the in vivo response to the drug(s)being tested than animal and other in-vitro models and so offer safer,and potentially cheaper, models for drug screening.

Finally, human ES cells could be used to develop new methods for geneticengineering. Currently, the genetic complement of mouse ES cells invitro can be modified easily by techniques such as homologousrecombination. Using this method, genes to direct differentiation to aspecific cell type or genes that express a desired protein product mightbe introduced into the ES cell line. Ultimately, if such techniquescould be developed using human ES cells, it may be possible to devisebetter methods for gene therapy.

At present the only source for embryonic stem cells is thepre-implantation blastocyst embryo.

The pluripotency of human post-implantation embryonic cells between thetime of implantation and the gastrulation process has as yet never beenexamined. Surani and Edwards teach in vitro culturing techniques ofhuman embryos to day 9, demonstrating the presence of proliferating andhealthy ICM [Edwards R. G., Surani M. A. H. (1978) Upsala Journal ofMedical Sciences 22: 39-50].

However, they did not examine the pluripotency of stem cells at thispost-implantation stage and did not isolate or culture them to allowtheir characterization.

Rathjen and colleagues teach a method for homogenous differentiation ofmouse embryonic stem cells into early primitive ectoderm-like (EPL)using conditioned medium of Hep G2 cells [Rathjen et al. 1999, J. ofCells Science 112, 601-612] and demonstrate some similarities betweenthem and the embryonic stem cells of the present invention. For exampleboth cell types have a colony morphology of epithelial-like structures,a higher tendency to differentiate into mesodermal tissues and a reducedability to either integrate into the embryonic germ layers afterinjection into mouse blastocysts [Lake et al. 2000, J. of Cells Science113, 555-566] or form teratomas. However, in sharp contrast to theembryonic stem cells of the present invention, the EPLs unique featurestogether with their ability to be cultured for limited passages in vitroare irreversible when the Hep G2 conditioned medium is removed [Rathjenet al. 1999 J. of Cells Science 112, 601-612; Lake et al. 2000, J. ofCells Science 113, 555-566]. The gene expression pattern of these cellsis also different. The EPL cells, for example, express brachyury only asearly EBs and not as undifferentiated cells like the embryonic stemcells of the present invention [Rathjen et al., 2000 J Cell Sci. 2000113:555-66]. Together with the fact that the isolated EPLs arenon-primate cells, the above mentioned differences indicate distinctcell populations.

The very broad range of potential applications for embryonic stem cellssuggests that the identification of additional sources together with thenovel stem cell lines derived therefrom, will be of critical importancefor medical research in general and the advancement of stem cellresearch in particular.

SUMMARY OF THE INVENTION

According to one aspect of the present invention there is provided anisolated primate embryonic cell characterized by expression of brachyuryand ability to differentiate to derivatives of each of an endoderm,mesoderm, and ectoderm tissue.

According to another aspect of the present invention there is provided acell culture comprising the isolated primate embryonic cellcharacterized by expression of brachyury and ability to differentiate toderivatives of each of an endoderm, mesoderm, and ectoderm tissue.

According to yet another aspect of the present invention there isprovided a method of generating a primate embryonic cell culturecomprising providing a blastocyst of at least nine days postfertilization, isolating cells from the blastocyst; and culturing theisolated cells, thereby generating the primate embryonic cell culture.

According to still another aspect of the present invention there isprovided a method of generating a primate embryonic cell line comprisingproviding a blastocyst of at least nine days post fertilization;isolating cells from the blastocyst; culturing the isolated cells; andcloning at least one of the isolated cells, thereby generating theprimate embryonic cell line.

According to an additional aspect of the present invention there isprovided a pharmaceutical composition comprising as an active ingredienta cell characterized by expression of brachyury and ability todifferentiate to derivatives of each of an endoderm, mesoderm, andectoderm tissue and a pharmaceutically acceptable carrier.

According to yet an additional aspect of the present invention there isprovided a method of treating or preventing a disease in which celltransplantation is therapeutically beneficial comprising inducing celldifferentiation in the isolated human embryonic cell characterized byexpression of brachyury and ability to differentiate to derivatives ofeach of an endoderm, mesoderm, and ectoderm tissue and transplanting atherapeutically effective amount of the differentiated cells into asubject in need thereof, thereby preventing or treating a disease inwhich cell transplantation is therapeutically beneficial.

According to still an additional aspect of the present invention thereis provided a method of generating a lineage specific cell comprisinginducing a lineage-specific cell differentiation in the isolated primateembryonic cell characterized by expression of brachyury and ability todifferentiate to derivatives of each of an endoderm, mesoderm, andectoderm tissue, thereby generating the lineage specific cell.

According to further features in preferred embodiments of the inventiondescribed below, the isolated primate embryonic cell further expressesat least one cartilage marker

According to still further features in the described preferredembodiments, the at least one cartilage marker is selected from thegroup consisting of COMP, aggrecan and collagen type II.

According to still further features in the described preferredembodiments, the cell culture further comprises feeder cells.

According to still further features in the described preferredembodiments, the cell culture, further comprises growth medium.

According to still further features in the described preferredembodiments, the providing the blastocyst is effected by ex-vivoculturing the blastocyst.

According to still further features in the described preferredembodiments, the ex-vivo culturing the blastocyst is effected on feedercells.

According to still further features in the described preferredembodiments, the ex-vivo culturing the blastocyst is effected on asynthetic surface.

According to still further features in the described preferredembodiments, the culturing the isolated cells is effected on feedercells.

According to still further features in the described preferredembodiments, the culturing the isolated cells is effected on a syntheticsurface.

According to still further features in the described preferredembodiments, the isolated primate embryonic cell maintains a stablenormal karotype for at least one year.

According to still further features in the described preferredembodiments, the isolated primate embryonic cell expresses SSEA4 andTRA-1-60 markers.

According to still further features in the described preferredembodiments, the isolated primate embryonic cell does not express SSEA1marker.

According to still further features in the described preferredembodiments, the isolated primate embryonic cell expresses less TRA-1-81marker than an embryonic stem cell of the same primate species notexpressing brachyury using identical assay conditions.

According to still further features in the described preferredembodiments, the isolated primate embryonic cell is capable of colonyorganization of columnar epithelium with villi throughout the upper sideof the colony.

According to still further features in the described preferredembodiments, the isolated primate embryonic cell has an OCT4 proteinlevel lower than the OCT4 protein level in an embryonic stem cell of thesame primate species not expressing brachycury using identical assayconditions.

According to still further features in the described preferredembodiments, the isolated primate embryonic cell expresses moremesodermal differentiating markers than an embryonic stem cell of anidentical primate not expressing brachyury using identical assayconditions.

According to still further features in the described preferredembodiments, the isolated primate cell is genetically modified.

According to still further features in the described preferredembodiments, the feeder cells are mouse feeder cells or human feedercells.

According to still further features in the described preferredembodiments, the mouse feeder cells are mitotically inactivated mouseembryonic fibroblasts or primary mouse embryonic fibroblasts.

According to still further features in the described preferredembodiments, the human feeder cells are selected from the groupcomprising embryonic fibroblast cells, adult fallopian epithelial cellsand foreskin cells.

According to still further features in the described preferredembodiments, the isolated primate embryonic cell is in anundifferentiated proliferative state for at least 100 passages.

According to still further features in the described preferredembodiments, the isolated primate embryonic cell is immortalized.

According to still further features in the described preferredembodiments, the primate is a human.

According to still further features in the described preferredembodiments, the method of treating or preventing a disease in whichcell transplantation is therapeutically beneficial further comprisesgenetically modifying the differentiated cell to express a therapeuticagent.

According to still further features in the described preferredembodiments, the inducing cell differentiation comprises geneticallymodifying a plurality of the cells.

The present invention successfully addresses the shortcomings of thepresently known configurations by providing a

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the patentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

In the drawings:

FIGS. 1A-E are photographs of light microscopy images depicting thegeneration of extended blastocyst cell lines (EBCs). FIGS. 1A-D depictthe generation of the J3 cell line. FIG. 1A depicts an early blastocystprior to plating on mouse embryonic feeder cells (MEFs). Hoffmanresolution bar=120 μM. FIG. 1B depicts the identical human embryofollowing plating for five days on MEFs. Hoffman resolution bar=120 μM.FIG. 1C depicts the identical human embryo following plating for ninedays on MEFs. Hoffman resolution bar=120 μM. FIG. 1D depicts theresulting EBC cells. Hoffman resolution bar=20 μM. FIG. 1E depicts anextended blastocyst cell line (J6) following a second passage for tendays on MEFs. The yellow line represents the outline of a condense areacontaining the stem cells. Hoffman resolution bar=135 μM.

FIGS. 2A-E are photographs of light (FIGS. 2A-C; Hoffman resolutionbar=20 μM; H&E staining) and electron (2D-E; Hoffman resolution bar=8μM) microscopy images depicting the organization of EBC and ESCcolonies. FIG. 2A depicts the organization of an ESC colony I6. FIG. 2Bdepicts the organization of the EBC colony J3. FIG. 2C depicts theorganization of the EBC colony J6. FIG. 2D depicts the organization ofthe pre-implantation cell line H9 as observed with electron microscopy.FIG. 2E depicts the organization of the EBC cell line J3 as observedwith electron microscopy.

FIGS. 3A-G are a series of photographs depicting the fluorescentimmunostaining with stage specific markers of EBCs and ESCs. FIG. 3Adepicts J3 colony staining with SSEA4. FIGS. 3B and 3C depict DM-1 (anhESC line derived from ICM) staining with SSEA4 under a light (FIG. 3B)and fluorescent (3C) microscope. FIGS. 3D and 3E depict DM-1 stainingwith TRA-1-6 under a light (FIG. 3D) and fluorescent (FIG. 3E)microscope. FIGS. 3F and 3G depict WS-1 (an hESC line derived from ICM)staining with TRA-1-81 under a light (FIG. 3F) and fluorescent (FIG. 3G)microscope. Phase contrast Bar=50 μM.

FIGS. 4A-B are photographs of teratoma sections stained withhematoxylin/eosin (H&E) as observed under a light microscope. Hoffmanresolution bar=20 μM. The teratomas were generated followingintroduction of cells from I5 (FIG. 4A) and J2 (FIG. 4B) cell lines intofour-week-old male SCID-beige mice.

FIGS. 5A-F are photographs of teratoma sections of EBC lines stainedwith H&E as observed under a light microscope demonstratingrepresentative tissues of the three embryonic germ layers. The teratomaswere generated following introduction of cells from J2 cell lines intofour-week-old male SCID-beige mice. FIG. 5A depicts epithelium. Hoffmanresolution bar=100 μM. FIG. 5B depicts secretory glands. Hoffmanresolution bar=40 μM. FIG. 5C depicts fat tissue. Hoffman resolutionbar=40 μM. FIG. 5D depicts mesenchymal tissue. Hoffman resolution bar=40μM. FIG. 5E depicts bone tissue. Hoffman resolution bar=40 μM. FIG. 5Fdepicts myelinated nerve. Hoffman resolution bar=60 μM.

FIG. 6 is a photograph of a teratoma section of an EBD line stained withH&E showing cartilage and bone tissue as observed under a lightmicroscope. The teratoma was generated following introduction of cellsfrom J6 cell lines into four-week-old male SCID-beige mice. Hoffmanresolution bar=20 μM.

FIG. 7 is a bar graph depicting the relative expression of Oct 4according to quantitative real-time PCR analysis in I3, J3 and J6 cellline. NTC=no template control, i.e. samples where no cDNA was added. I3was used as the calibrator=1 (100%).

FIGS. 8A-D are dot graphs and photographs from chip DNA analysis of theGEarray S series stem cells genes, Hs601.2. FIG. 8A is a dot graphcomparing the gene expressions of two different hESC lines. FIG. 8B is adot graph comparing the gene expressions of two different EBC celllines. FIG. 8C is a dot graph comparing the average gene expression ofhESC with EBC cell lines. FIG. 8D is a representative example of thechip.

FIGS. 9A-I are photographs of the J6 undifferentiated EBC cell linedouble stained with brachyury and Oct-4. FIGS. 9A, 9D and 9G depictbrachyury staining alone. FIGS. 9C, 9F and 9I depict Oct-4 stainingalone and FIGS. 9B, 9E and 9H depict the combined staining of bothbrachyury and Oct-4. Phase contrast bar=20 μM.

FIGS. 10A-I are photographs of the I3 hESC cell line double stained withbrachyury and Oct-4. FIGS. 10A, 10D and 10G depict brachyury stainingalone. FIGS. 10C, 10F and 10I depict Oct-4 staining alone and FIGS. 10B,10E and 10H depict the combined staining of both brachyury and Oct-4.FIGS. 10A-C are of a differentiating colony of hESC lines; FIGS. 10D-Fare of an undifferentiated colony of hESC line; FIGS. 10G-I are of anundifferentiated colony of hESC line. Phase contrast bar=20 μM.

FIGS. 11A-B are computer generated images following bioinformaticanalysis using the MAS 5.0 Affymetrix array analysis software of fourAffymetric focus gene chips hybridized with cDNA from four cell lines(I3, I6 J3 and J6). FIG. 11A is a dendrogram based on the similaritybetween the four tested lines (I3, I6 J3 and J6). The followingcalculation settings were used: Columns included: J6 Signal, Z Scores,I3 Signal, Z Scores, I6 Signal, Z Scores, J3 Signal, Z Scores; Emptyvalues replaced by: 0; Clustering method: Complete linkage (maximum);Similarity measure: Correlation; Ordering function: Average value. FIG.11B is a heat map of the 238 probe sets which were significantlydifferent in I cell lines and the J cell lines with a maximum p-value of0.05.

FIG. 12 is a pie chart illustrating the results obtained followingGo-Chat analysis of the 238 probe sets which were significantlydifferent in I cell lines and the J cell lines with a maximum p-value of0.05.

FIGS. 13A-F are photographs of two-dimensional gel electrophoresis of anarea within the gel that clearly differs between 5-day old EBs andundifferentiated hESCs. All treatments were performed on cells from theI4 cell line. FIG. 13A is a photograph of the Master gel (i.e. a gel onwhich MEF protein extract alone has been run) following two-dimensionalgel electrophoresis. FIG. 13B is a photograph of two-dimensional gelelectrophoresis of MEFs used to culture the hESCs. FIG. 13C is aduplicate of 13B. FIG. 13D is a photograph of two-dimensional gelelectrophoresis of undifferentiated hESCs. FIG. 13E is a photograph oftwo-dimensional gel electrophoresis of hESCs that were cultured for 10days as monolayers. FIG. 13F is a photograph of two-dimensional gelelectrophoresis of 5-days old EBs.

FIGS. 14A-F are photographs of two-dimensional gel electrophoresisdemonstrating an area within the gel that clearly differs betweendifferentiated hESCs and undifferentiated hESCs. All treatments areperformed on cells from the I4 cell line. FIG. 14A is a photograph ofthe Master gel following two-dimensional gel electrophoresis. FIG. 14Bis a photograph of two-dimensional gel electrophoresis of MEFs used toculture the hESCs. FIG. 13C is a duplicate of 14B. FIG. 14D is aphotograph of two-dimensional gel electrophoresis of undifferentiatedhESCs. FIG. 14E is a photograph of two-dimensional gel electrophoresisof hESCs that were cultured for 10 days as monolayers. FIG. 14F is aphotograph of two-dimensional gel electrophoresis of differentiatedhESCs.

FIGS. 15A-B are photographs of Alizarin Red stained EB derived cells(EBDs). FIG. 15A depicts EBDs resulted from I4 (hESC) cells and FIG. 15Bdepicts EBDs from J6 cells (DBC) treated with the same mediumsupplemented with TGF_(β3). While the hESC demonstrated no positivestaining the DBC cells demonstrated large area of clear red staining.FIG. 15A: Bar=15 μM and FIG. 15B: Bar=50 μM.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of isolated primate embryonic stem cells andmethods of generating stem cell cultures and cell lines therefrom.Specifically, the present invention relates to isolated embryonic stemcells which are characterized by expression of brachyury and theirability to differentiate to derivatives of endoderm, mesoderm, andectoderm tissues.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details set forth in the following description or exemplified bythe Examples. The invention is capable of other embodiments or of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein is for the purposeof description and should not be regarded as limiting.

Embryonic stem cells are characterized by their ability to propagateindefinitely in culture, as undifferentiated cells, while they can beinduced to differentiate in vivo into teratomas when injected into SClDmice [Thomson, J. A., et al., (1998), Science, 282, 1145-1147;Reubinoff, B. E., et al., (2000), Nature Biotechnol., 18, 399-404]. Theymay also differentiate in vitro into embryoid bodies (EBs) that containembryonic cells from the three germ layers (endoderm, mesoderm, andectoderm) [Itskovitz-Eldor, J. et al., Mol. Med., 6, 88-95]. Moreover,this differentiation can be somewhat directed by the addition of growthfactors into the culture media [Schuldiner, M et al., (2000) P.N.A.S.USA, 97, 11307-11312]. For example, ES cells can be induced todifferentiate in vitro into cardiomyocytes [Paquin et al., Proc. Nat.Acad. Sci. (2002) 99:9550-9555], hematopoietic cells [Weiss et al.,Hematol. Oncol. Clin. N. Amer. (1997) 11(6):1185-98; U.S. Pat. No.6,280,718], insulin-secreting beta cells [Assady et al., Diabetes (2001)50(8):1691-1697], and neural progenitors capable of differentiating intoastrocytes, oligodendrocytes, and mature neurons [Reubinoff et al.,Nature Biotechnology (2001) 19:1134-1140; U.S. Pat. No. 5,851,832].

Human ES cells may also be genetically manipulated in culture [Eiges etal., (2001) Curr. Biol., 11, 514-518] and the transfected cells remainpluripotent (i.e., capable through its progeny of giving rise in vivo toall the cell types which comprise the adult animal including the germcells) and retain a normal karyotype [Shuldiner et al., (2003) StemCells 21, 257-265].

As a result of their unique features, it has been suggested that humanES cells hold the promise of changing the face of cell transplantation,by replacing or restoring tissue that has been damaged by disease orinjury. Replacement of non-functional cells using ES cells technologycan offer a lifelong treatment. Thus, diseases that might be treated bytransplanting human ES-derived cells include Parkinson's disease,diabetes, traumatic spinal cord injury, Purkinje cell degeneration,Duchenne's muscular dystrophy, heart failure, and osteogenesisimperfecta.

ESCs have been successfully isolated from primates (Thomson et al., 1998Science, 282, 1145-1147; Amit et al., 2000 Dev. Biol. 227: 271-8), mice(Mills and Bradley, 2001, Trends Genet. 17: 331-9) and other specieswhere they are derived from the inner cell mass (ICM) of the mammalianblastocyst prior to the implantation stage.

Whilst reducing the present invention to practice, the present inventorshave uncovered that human embryonic stem cells may be derived from apost-implantation stage blastocyst. Moreover these stem cells displaydifferent characteristics to the traditional embryonic stem cellsderived from pre-implantation stage blastocysts while are still capableof differentiating to all the cell types which comprise the adult animal(Example 3, FIGS. 4A-B and 5A-F) and therefore represent a novel classof embryonic stem cell.

Thus, as described in the Examples section which follows embryonic stemcells derived from post-implantation/pre-gastrulation stage blastocysts[referred to herein as extended blastocyst cells (EBCs)] display adifferent colony morphology (FIGS. 2A-E) and express different levels ofboth cell surface markers and differentiation markers to ESCs derivedfrom pre-implantation stage blastocysts. Specifically, EBCs express manyearly differentiation markers, mostly mesodermal, as demonstrated byRT-PCR (Table 3, Example 3 and FIG. 7), histochemistry (FIGS. 3A-X, 9A-Iand 10A-I), two-dimensional gel electrophoresis (FIGS. 13A-F and 14A-F)and chip DNA analysis (FIGS. 8A-D, 11A-B and 12).

It will be appreciated that although ex vivo culturing of human embryosuntil day 9 has been previously reported, the prior art does not teachof stem cell isolation or culturing. Moreover, the pluripotency of thesestem cells at this post-implantation stage was not examined [Edwards R.G., Surani M. A. H. (1978) Upsala Journal of Medical Sciences 22:39-50].

Thus, according to one aspect of the present invention there is providedan isolated primate embryonic cell characterized by expression ofbrachyury and ability to differentiate to derivatives of each of anendoderm, mesoderm, and ectoderm tissue.

As used herein the phrase “primate embryonic cell” refers to a cell fromembryonic primate origin. Typically the embryonic cell of this aspect ofthe present invention has a high nuclear/cytoplasmic ratio and prominentnucleolus.

As used herein the term “primate” refers to both higher and lowerprimates. Preferably the primate is a human.

According to this aspect of the present invention, the term “isolated”refers to an embryonic cell or a plurality of embryonic cells that havebeen removed from their naturally-occurring in-vivo environment (i.e.post-implantation/pre-gastrulation stage blastocyst). Preferably theisolated embryonic cell of this aspect of the present invention (i.e.,expressing Brachyury and being capable of differentiating to derivativesof each of an endoderm, mesoderm, and ectoderm tissue) is substantiallyfree from other substances (e.g., other cells, proteins, nucleic acids,etc.) that are present in its in-vivo environment. Methods for removingembryonic cells from a post-implantation/pre-gastrulation stageblastocyst are described hereinbelow and in the Examples section whichfollows.

As used herein, the term “brachyury” refers to the human T-boxtranscription factor, of Swiss Prot No. 015178 and homologues andorthologues thereof, such as encoded by the polynucleotide sequenceslisted in Table 1 below.

TABLE 1 Species GenBank Accession Number Chimpanzee 472186 ChimpanzeeXM_527563.1 Chimpanzee XP_527563.1

As used herein, the phrase “expression of brachyury” refers tomRNA/protein expression of brachyury. Preferably, the endogenousexpression of Brachyury in the isolated cells of the present inventionis at least two times higher, preferably at least five times higher andmore preferably at least ten times higher than the endogenous expressionof Brachyury protein in embryonic stem cells derived frompre-implantation blastocysts. Increased expression of Brachyury proteinmay be confirmed using standard techniques known in the art furtherdescribed hereinbelow.

As used herein, the phrase “derivatives of each of an endoderm, mesodermand ectoderm tissue” encompasses fully or partially differentiatedcells. This characteristic of the cells of the present inventionresembles the ability of embryonic stem cells to derivatives of each ofan endoderm, mesoderm and ectoderm tissue. Examples of endodermderivatives include, but are not limited to, hepatocytes and pancreaticcells. Examples of mesoderm derivatives, include but are not limited to,osseous, cartilaginous, elastic, fibrous connective tissues, myocytes,myocardial cells, bone marrow cells, vascular cells (namely endothelialand smooth muscle cells), and hematopoietic cells. Examples of ectodermderivatives include but are not limited to neural, retina and epidermalcells.

The isolated primate embryonic cells of the present invention may beobtained from a blastocyst of at least nine days post fertilization at astage prior to gastrulation. Blastocysts less than nine days old may beobtained from in vivo preimplantation embryos, in vitro fertilized (IVF)embryos or from single cell embryos expanded to the blastocyst stage.Prior to culturing the blastocyst, the zona pellucida is digested (forexample by Tyrode's acidic solution (Sigma Aldrich, St Louis, Mo., USA))so as to expose the inner cell mass. The blastocysts are then culturedas whole embryos for at least nine and no more than fourteen days postfertilization (i.e. prior to the gastrulation event) in vitro usingstandard embryonic stem cell culturing methods.

Blastocyst culturing may be effected using currently practiced ESculturing methods. These are mainly based on the use of feeder celllayers which secrete factors needed for stem cell proliferation, whileat the same time, inhibit their differentiation. Feeder cell-freesystems have also been used in ES cell culturing, such systems utilizematrices supplemented with serum, cytokines and growth factors as areplacement for the feeder cell layer. The following summarizesculturing techniques which may be used in each of the steps ofgenerating the embryonic cells of the present invention (also referredto as embryonic stem cells of the present invention). Currentlypreferred configuration of blastocyst culturing is described in Example1 of the Examples section which follows.

Feeder-Layer Based Cultures

Mouse Feeder Layers—

The most common method for culturing ES cells is based on mouseembryonic fibroblasts (MEF) as a feeder cell layer supplemented withtissue culture medium containing serum or leukemia inhibitor factor(LIF) which supports the proliferation and the pluripotency of the EScells [Thomson J A, Itskovitz-Eldor J, Shapiro S S, Waknitz M A,Swiergiel J J, Marshall V S, Jones J M. (1998). Embryonic stem celllines derived from human blastocysts. Science 282: 1145-7; Reubinoff BE, Pera M F, Fong C, Trounson A, Bongso A. (2000). Embryonic stem celllines from human blastocysts: somatic differentiation in vitro. Nat.Biotechnol. 18: 399-404]. MEF cells are derived from day 12-13 mouseembryos in medium supplemented with fetal bovine serum. Under theseconditions mouse ES cells can be maintained in culture as pluripotentstem cells, preserving their phenotypical and functionalcharacteristics. However, unlike mouse ES cells, the presence ofexogenously added LIF does not prevent differentiation of human ES cells[Thomson J A, Itskovitz-Eldor J, Shapiro S S, Waknitz M A, Swiergiel JJ, Marshall V S, Jones J M. (1998). Embryonic stem cell lines derivedfrom human blastocysts. Science 282: 1145-7; Reubinoff B E, Pera M F,Fong C, Trounson A, Bongso A. (2000). Embryonic stem cell lines fromhuman blastocysts: somatic differentiation in vitro. Nat. Biotechnol.18: 399-404]. Furthermore, the use of feeder cells substantiallyincreases the cost of production, and makes scale-up of human ES cellculture impractical. Additionally, the feeder cells are metabolicallyinactivated to keep them from outgrowing the stem cells; hence it isnecessary to have fresh feeder cells for each splitting of human ESculture. Since at present, the separation of feeder cell components fromembryonic cells prepared in bulk culture cannot be efficiently achieved,feeder cell layer-prepared ES cultures are not suitable for humantherapy.

ES cells can also be cultured on MEF under serum-free conditions usingserum replacement supplemented with basic fibroblast growth factor(bFGF) [Amit M, Carpenter M K, Inokuma M S, Chiu C P, Harris C P,Waknitz M A, Itskovitz-Eldor J, Thomson J A. (2000). Clonally derivedhuman embryonic stem cell lines maintain pluripotency and proliferativepotential for prolonged periods of culture. Dev. Biol. 227: 271-8].Under these conditions the cloning efficiency of ES cells is 4 timeshigher than under fetal bovine serum. In addition, following 6 months ofculturing under serum replacement the ES cells still maintain theirpluripotency as indicated by their ability to form teratomas whichcontain all three embryonic germ layers. Although this system uses abetter-defined culture conditions, the presence of mouse cells in theculture exposes the human culture to pathogens which restricts their usein cell-based therapy.

Human Embryonic Fibroblasts or Adult Fallopian Epithelial Cells asFeeder Cell Layers—

Human ES cells can be grown and maintained using human embryonicfibroblasts or adult fallopian epithelial cells. When grown on thesehuman feeder cells the human ES cells exhibit normal karyotypes, presentalkaline phosphatase activity, express Oct-4 and other embryonic cellsurface markers including SSEA-3, SSEA-4, TRA-1-60, and GCTM-2, formteratomas in vivo, and retain all key morphological characteristics[Richards M, Fong C Y, Chan W K, Wong P C, Bongso A. (2002). Humanfeeders support prolonged undifferentiated growth of human inner cellmasses and embryonic stem cells. Nat. Biotechnol. 20: 933-6]. However,the major disadvantage of using human embryonic fibroblasts or adultfallopian tube epithelial cells as feeder cells is that both of thesecell lines have a limited passage capacity of only 8-10 times, therebylimiting the ability of a prolonged ES growth period. For a prolongedculturing period, the ES cells must be grown on human feeder cellsoriginated from several subjects which results in an increasedvariability in culture conditions.

Foreskin Feeder Layers—

Human ES cells can be cultured on human foreskin feeder layer asdisclosed in U.S. patent application Ser. No. 10/368,045. Foreskinderived feeder cell layers consist of a complete animal-free environmentsuitable for culturing human ES cells. In addition, foreskin cells canbe maintained in culture for as long as 42 passages since theirderivation, providing the ES cells with a relatively constantenvironment. Under these conditions the human ES cells were found to befunctionally indistinct from cells grown with alternate protocols (e.g.,MEF). Following differentiation, ES cells expressed genes associatedwith all three embryonal germ layers, in vitro, and formed teratomas invivo, consisting of tissue arising from all three germ layers. Inaddition, unlike human fallopian epithelial cells or human embryonicfibroblasts, human ES cells cultured on foreskin feeder layers weremaintained in culture in a pluripotent and undifferentiated state for atleast 87 passages. However, although foreskin cells can be maintained inculture for long periods (i.e., 42 passages), the foreskin culturesystem is not well-defined due to differences between separate batches.In addition, human feeder layer-based culture systems would stillrequire the simultaneous growth of both feeder layers and hES cells.Therefore, feeder-free culturing systems have been developed.

Feeder-Free Cultures

Stem cells can be grown on a solid surface such as an extracellularmatrix (e.g., Matrigel® or laminin) in the presence of a culture medium.Unlike feeder-based cultures which require the simultaneous growth offeeder cells and stem cells and which may result in mixed cellpopulations, stem cells grown on feeder-free systems are easilyseparated from the surface. The culture medium used for growing the stemcells contains factors that effectively inhibit differentiation andpromote their growth such as MEF-conditioned medium and bFGF. However,commonly used feeder-free culturing systems utilize an animal-basedmatrix (e.g., Matrigel®) supplemented with mouse or bovine serum, orwith MEF conditioned medium [Xu C, et al. (2001). Feeder-free growth ofundifferentiated human embryonic stem cells. Nat Biotechnol. 19: 971-4]which present the risk of animal pathogen cross-transfer to the human EScells, thus compromising future clinical applications.

The embryonic stem cells of the present invention can be isolated byvirtue of their morphology (e.g. small cell with large nucleus) and/orby any other stem cell distinguishing feature. Isolation may be achievedby chemical or mechanical means or both. Preferably mechanical isolationand removal by a micropipette is used. Mechanical isolation may becombined with a chemical or enzymatic treatment to aid with dissociationof the cells, such as Ca²⁺/Mg²⁺ free PBS medium or dispase.

The isolated embryonic stem cells of this invention may be cultured inany way which ensures their non-differentiated state as describedhereinabove. An exemplary culturing medium comprises 85% ko-DMEM andsupplemented with 15% ko-serum replacement, 1 mM L-glutamine, 0.1 mMβ-mercaptoethanol, 1% non-essential amino acid stock and 4 ng/ml basicfibroblast growth factor (bFGF) (all products from Gibco Invitrogencorporation products, San Diago, Calif., USA). Methods of qualifying theembryonic stem cells of the present invention are well known in the art.For example, differentiation state may be determined by injecting cellsinto a 8-12 week old SCID mice (FIGS. 5A-F) as described by Evans etal., [Evans M J and Kaufman M (1983) Cancer Surv. 2: 185-208], whichupon injection form teratomas. Teratomas are typically fixed using 4%paraformaldehyde and histologically examined for the three germ layers.Alternatively, this can be confirmed by determining their ability toform embryonal bodies as described in Example 3 of the Examples sectionwhich follows.

The embryonic stem cells of the present invention can also be qualifiedby the expression of cell markers. Typically, expression of cell markerscan be detected using a variety of cell biology, molecular biology andbiochemical techniques which are well known in the art. For example flowcytometry may be used to detect membrane-bound markers,immunohistochemistry may be used to detect intracellular markers, andenzyme-linked immunoassays may be used to detect markers secreted intothe medium. The expression of protein markers can also be detected atthe mRNA level by reverse transcriptase-PCR (using marker-specificprimers), Northern blots and oligonucleotide microarrays.

Thus, the embryonic stem cells of the present invention may be qualifiedby the expression of stage-specific embryonic antigens (SSEA).Antibodies for SSEA markers are available from the Developmental StudiesHybridoma Bank (Bethesda Md.). Other markers which are typically usedfor the qualification of embryonic stem cells are Tra-1-60 and Tra-1-81(antibodies available from Chemicon International, Temecula, Canada) andOctamer binding transcription factor 4 (OCT-4) (Santa Cruz). OCT-4(GenBank Accession No. NM_(—)00270) is a member of the POU family oftranscription factors. OCT-4 transcription is activated between the 4and 80 cell stage in the developing embryo, and it is highly expressedin the expanding blastocyst and then in the pluripotent cells of the eggcylinder. Transcription is down-regulated as the primitive ectodermdifferentiates to form mesoderm, and by 8.5 days post coitum isrestricted to migrating primordial germ cells. Similar to embryonic stemcells not expressing brachyury, the embryonic stem cells of the presentinvention express SSEA4 and TRA-1-60 markers. However, in contrast toembryonic stem cells not expressing brachyury, the embryonic stem cellsof the present invention do not express SSEA1 marker. In addition, theembryonic stem cells of the present invention express less TRA-1-81 andOCT-4 marker than embryonic stem cells not expressing brachyury usingidentical assay conditions.

The isolated primate embryonic stem cell of the present inventiontypically expresses more mesodermal differentiating markers than anembryonic stem cell of an identical primate not expressing brachyury.Typically, the isolated primate embryonic stem cells of the presentinvention express cartilage markers. Examples of cartilage markers whichare expressed by the cells of the present invention include but are notlimited to COMP (GenBank Accession No. L32137), aggrecan (GenBankAccession No. X17406) and collagen type II (GenBank Accession No.X06268).

Examples of other mesodermal differentiating markers that are expressedat a higher level in the embryonic stem cells of the present inventioninclude, but are not limited to cartilage link protein (GenBankAccession No. U43328), cardiac actin (GenBank Accession No.NM_(—)005159), BMP4 (GenBank Accession No. D30751) and BMP2 (GenBankAccession No. NM_(—)001200).

Another method of qualification of embryonic stem cells is based ongenetic analysis. Karotyping is one type of morphological analysisroutinely performed for the qualification of embryonic stem cells. It isimportant in order to verify cytological euploidity, wherein allchromosomes are present and not detectably altered during culturing.Cultured stem cells can be karyotyped using a standard Giemsa stainingand compared to published karyotypes of the corresponding species.

As is illustrated in Example 2 of the Examples section below, embryonicstem cells of the present invention retain a normal karyotype followingat least twenty passages.

Microscopy is a type of morphological analysis routinely performed forthe qualification of embryonic stem cells.

As demonstrated in Example 2 (FIGS. 2A-E) the isolated primate embryonicstem cells of the present invention are capable of a colony organizationof columnar epithelium with villi throughout the upper side of thecolony. Typically the colonies of embryonic stem cells not expressingbrachyury are organized as stratified epithelium, with no villi facingthe upper side (FIG. 2A). Furthermore, electron microscopy indicatedthat zonula occludente junctions, typical for columnar epithelium, couldbe detected between the embryonic stem cells of the present inventionand not between the upper rows of embryonic stem cells not expressingbrachyury (FIGS. 2D-E).

Preferably, the embryonic stem cells of the present invention remain inan undifferentiated proliferative state for at least 20, 30, 40, 50, 60,70, 80, 90, preferably at least one hundred passages.

Stem cells generated according to the teachings of the present inventionmay be cultured on feeder cells or in a xenofree environment such as inthe presence of synthetic extracellular matrix components (e.g.,Matrigel™) as described above.

An isolated embryonic stem cell may be cloned from the culture describedhereinabove in order to generate an embryonic stem cell line. Methods ofcloning are well known in the art.

Cell lines of the present invention can be produced by immortalizing thecloned cells by methods known in the art, including, for example,expressing a telomerase gene in the cells (Wei, W. et al., 2003.Abolition of Cyclin-Dependent Kinase Inhibitor p16Ink4a and p21Cip1/Waf1Functions Permits Ras-Induced Anchorage-Independent Growth inTelomerase-Immortalized Human Fibroblasts. Mol Cell Biol. 23: 2859-2870)or co-culturing the cells with NIH 3T3 hph-HOX11 retroviral producercells (Hawley, R. G. et al., 1994. The HOX11 homeobox-containing gene ofhuman leukemia immortalizes murine hematopoietic precursors. Oncogene 9:1-12).

The embryonic stem cells may be further genetically modified at anystage of isolation. For example, they may be genetically modifiedthrough introduction of vectors expressing a selectable marker under thecontrol of a stem cell specific promoter such as Oct-4. Somedifferentiated progeny of embryonic stem cells may produce productswhich are inhibitory to stem cell renewal or survival. Thereforeselection against such differentiated cells, facilitated by theintroduction of a construct such as that described above, may promotestem cell growth and prevent differentiation. The stem cells may begenetically modified at any stage with markers so that the markers arecarried through to any stage of cultivation. The markers may be used topurify the differentiated or undifferentiated stem cell population atany stage of cultivation.

Alternatively, the embryonic stem cells of the present invention may begenetically modified to express a therapeutic agent or to directdifferentiation into a specific cell lineage.

Various methods can be used to introduce an exogenous gene (e.g. thetelomerase gene) in primate embryonic stem cells. Such methods aregenerally described in Sambrook et al., Molecular Cloning: A LaboratoryManual, Cold Springs Harbor Laboratory, New York (1989, 1992), inAusubel et al., Current Protocols in Molecular Biology, John Wiley andSons, Baltimore, Md. (1989), Chang et al., Somatic Gene Therapy, CRCPress, Ann Arbor, Mich. (1995), Vega et al., Gene Targeting, CRC Press,Ann Arbor Mich. (1995), Vectors: A Survey of Molecular Cloning Vectorsand Their Uses, Butterworths, Boston Mass. (1988) and Gilboa et al.[Biotechniques 4 (6): 504-512, 1986] and include, for example, stable ortransient transfection, lipofection, electroporation and infection withrecombinant viral vectors. In addition, see U.S. Pat. Nos. 5,464,764 and5,487,992 for positive-negative selection methods.

Introduction of nucleic acids by viral infection offers severaladvantages over other methods such as lipofection and electroporation,since higher transfection efficiency can be obtained due to theinfectious nature of viruses.

The embryonic stem cells derived according to the teachings of thepresent invention can be used for several commercial and researchapplications.

Cultured embryonic stem cells of the present invention can bedifferentiated into restricted developmental lineage cells, orterminally differentiated cells.

Differentiation of stem cells can be initiated by allowing overgrowth ofundifferentiated human ES cells in suspension culture forming embryoidbodies or by plating ES cells under conditions that promotedifferentiation in a particular manner. Such conditions may includewithdrawing or adding nutrients, growth factors or cytokines to themedium, changing the oxygen pressure, or altering the substrate on theculture surface. For example, embryonic stem cells can be induced todifferentiate in vitro into cardiomyocytes [Paquin et al., Proc. Nat.Acad. Sci. (2002) 99:9550-9555]. Several factors alone or in combinationhave been shown to enrich cardiac differentiation such as hepatocytegrowth factor (HGF), epidermal growth factor (EGF), basic fibroblastgrowth factor (bFGF), transforming growth factor β1 (TGF β1), plateletderived growth factor (PDGF), sphingosine-1-phosphate, retinoic acid,5-azacytidine and vitamin C. Embryonic stem cells have also been inducedto differentiate into neural or glial lineages [Reubinoff et al., NatureBiotechnology (2001) 19:1134-1140; U.S. Pat. No. 5,851,832]. For theirgeneration, the medium typically includes any of the following factorsor medium constituents in an effective combination: Brain derivedneurotrophic factor (BDNF), neutrotrophin-3 (NT-3), NT-4, epidermalgrowth factor (EGF), ciliary neurotrophic factor (CNTF), nerve growthfactor (NGF), retinoic acid (RA), sonic hedgehog, FGF-8, ascorbic acid,forskolin, fetal bovine serum (FBS), and bone morphogenic proteins(BMPs). Embryonic stem cells have also been induced to differentiateinto hematopoietic cells [Weiss et al., Hematol. Oncol. Clin. N. Amer.(1997) 11(6):1185-98; U.S. Pat. No. 6,280,718] and insulin-secretingbeta cells [Assady et al., Diabetes (2001) 50(8):1691-1697].

Differentiation of stem cells can also be directed by geneticmodification. Several transcription factors have been demonstrated toregulate differentiation of ES cells to specific cell types[Levinson-Dushnik M., Benvenisty N., Cell Biol. 17: 3817-3822, 1997].Ectopic over-expression of such factors stimulates ES cells todifferentiate selectively into certain cell types. For exampleover-expression of the transcription factor GATA-4 was shown to inducecardiomyocyte differentiation [Grepin C., et al., Development 124:2387-2395, 1997; Fujikura J., et al., Genes Dev. 16: 784-789, 2002;Kanda S., et al., Hepatol. Res. 26:225-231, 2003]. Techniques for thegenetic modification of the present invention are described hereinabove.

Alternatively, the embryonic stem cells of the present invention may beseeded over a porous scaffold (e.g. an alignate scaffold) as describedin U.S. Pat. Appl. No. 60/604,002 to allow the formation of embryoidbodies (EBs) thereby allowing partial differentiation. The threedimensional scaffolds may be coated with components of extracellularmatrix such as fibronectin, laminin, collagen and/or supplemented withcytokines, growth factors and chemokines. Cell seeding is effected in amanner which enables even distribution of the cells on/within thescaffold. One approach which can be utilized to achieve evendistribution is seeding under a centrifugal force (Dar et al., 2002,Biotechnol Bioeng 80:305-312). Cells are preferably seeded at aconcentration which ensures entrapment within the scaffold and maximalformation of EBs preferably about 5×10⁷ cells per cm³ scaffold. Theembryoid bodies may be further differentiated along specific celllineages as described hereinabove.

Since the cells of the present invention may be differentiated in alineage specific fashion, they may be used for human cell-based therapyand tissue regeneration.

Thus, according to another aspect of the present invention there isprovided a method of treating a disease in which cell transplantation istherapeutically beneficial.

The method according to this aspect of the present invention is effectedby inducing cell differentiation in the isolated human embryonic stemcell of the present invention and transplanting a therapeuticallyeffective amount of the differentiated cells into a subject in needthereof, thereby preventing or treating a disease in which celltransplantation is therapeutically beneficial.

As used herein “a disease in which cell transplantation istherapeutically beneficial” refers a disease, conditions or disordersuch as a neurological disorder (e.g. Parkinson's disease), a musculardisorder (e.g. muscular dystrophy), a cardiovascular disorder (e.g.heart failure), an hematological disorder (e.g. leukemia, lymphoma,thalassemia and sickle cell anemia), a metabolic disorder (e.g. Type Idiabetes), a skin disorder (e.g. psoriasis), a liver disorder (e.g.acute liver failure) that may be treated by cell transplantation.

The phrase “treating” refers to inhibiting or arresting the developmentof a disease, disorder or condition and/or causing the reduction,remission, or regression of a disease, disorder or condition in anindividual suffering from, or diagnosed with, the disease, disorder orcondition. Those of skill in the art will be aware of variousmethodologies and assays which can be used to assess the development ofa disease, disorder or condition, and similarly, various methodologiesand assays which can be used to assess the reduction, remission orregression of a disease, disorder or condition.

As used herein, “transplanting” refers to a means for providing thedifferentiated embryonic stem cells of the present invention, using anysuitable route, e.g., oral, sublingual intravenous, subcutaneous,transcutaneous, intramuscular, intracutaneous, intrathecal, intraperitoneal, intra spleenic, intra hepatic, intra pancreatic, intracardiac, epidural, intraoccular, intracranial, inhalation, rectal,vaginal, and the like. Cells may be transplanted as are or attached to ascaffold (matrix). Differentiation of the stem cells of the presentinvention may require specific environments for differentiation, whichinclude the presence of a supporting scaffold (e.g. osteoblasts) The 3Dscaffold provides a supporting frame and may act as a template forosteogenesis.

Differentiated embryonic stem cells of the present invention can beutilized in treating various disorders. For example, oligodendrocyteprecursors can be used to treat myelin disorders (Repair of myelindisease: Strategies and progress in animal models. Molecular MedicineToday. 1997, 554-561), chondrocytes or mesenchymal cells can be used intreatment of bone and cartilage defects (U.S. Pat. No. 4,642,120) andcells of the epithelial lineage can be used in skin regeneration of awound or burn (U.S. Pat. No. 5,716,411).

For certain disorders, such as genetic disorders in which a specificgene product is missing [e.g., lack of the CFTR gene-product in cysticfibrosis patients (Davies J C, 2002. New therapeutic approaches forcystic fibrosis lung disease. J. R. Soc. Med. 95 Suppl 41:58-67)], theembryonic stem cells of the present invention are preferably manipulatedto over-express the mutated gene prior to their administration to theindividual. It will be appreciated that for other disorders, the ESCs ofthe present invention should be manipulated to exclude certain genes.

Preferably, the embryonic stem cells of the present invention are atleast partially differentiated prior to transformation as implantationof undifferentiated ES cells may lead to formation of benign teratomasin the recipients. This may be achieved by designing a transgenicmethodology to eliminate residual minority stem cells fromdifferentiated ES cell cultures, for example based on negative selectionof Oct 4-expressing cells. Use of a positive selection transgene toachieve lineage-directed differentiation would also reduce the risk oftumor formation by selecting against the remaining undifferentiated,proliferating stem cell population.

The embryonic stem cells of the present invention may be geneticallyengineered (such as by using the above teachings) to reduce or eliminateimmune-mediated rejection so that lifelong pharmacologicimmunosuppression would not be required.

Homologous recombination has been used to “knock-out” majorhistocompatibility complex (MHC) class I and class II molecules in mouseES cells [Grusby M J, et al., Proc Natl Acad Sci USA 1993;90:3913-3917]. However, MHC class I- and class II-deficient skin graftsare still rejected, possibly on the basis of indirectallo-recognition-mediated rejection and/or natural killer cell-mediateddestruction [Grusby M J, et al., Proc Natl Acad Sci USA 1993;90:3913-3917]. Thus, in addition to deleting foreign MHC genes, desiredMHC genes may also be “knocked-in”, so that ES cell-derivativetransplants of the present invention are seen as “self” by theprospective recipient [Westphal C H, Leder P. Curr Biol 1997;7:530-533]. Alternatively, genes for immunosuppressive molecules such asFas-ligand could be inserted into the ES cells of the present invention,or important immune-stimulating proteins, such as B7 antigens orCD40-ligand, could be deleted from ES cells [Harlan D M, Kirk A D, JAMA1999; 282:1076-1082]. Irrespective of the method used, the ability tostably integrate genetic modifications into ES cells provides anadvantage over using adult somatic cells, which are less reliablygenetically altered.

Nuclear transfer technology may provide a more precise means to preventrejection of the transplanted ES cells of the present invention. Thistechnique would lead to ES cell-derived cells that are an exact geneticmatch to the recipient. In this way, there would be minimal host immuneresponse since all nuclear genes, including major and minorhistocompatibility loci, would be seen as “self.”

In this technique, a nucleus is extracted from a normal somatic cell ofa patient, e.g. from a skin biopsy, and then injected into an enucleatedoocyte. Oocyte cytoplasm has the ability to reprogram differentiatednuclei, and as such, would reestablish an embryonic gene expressionprogram in the chromatin of the somatic cell nucleus. A delayedblastocyst developing from this oocyte would be a source for thederivation of a new ES cell line of the present invention, which wouldbe genetically matched for each nuclear gene of the patient. In thissetting, the potential immune-mediated destruction of the graft would belimited to minor antigen differences derived from mitochondrial genes orto autoimmune processes, such as diabetes. Combining nuclear transfertechnology and ES cell derivation has been successfully achieved in cowsand mice, in order to establish transgenic ES cell lines fromreprogrammed somatic cell nuclei [First NL, Thomson J., Nat Biotechnol1998; 16:620-621].

Establishing hematopoietic chimerism is another potential means ofpreventing rejection of the transplanted cells of the present invention.By using the same ES cell lines to derive both hematopoietic stem cellsand other lineages, it may be possible to initially achievehematopoietic chimerism followed by engraftment of a second cell type.The second lineage would not be rejected as it would be regarded as“self” by the chimeric patient's bone marrow and immune system, whichwere derived, in part, from the same ES cell line. No long-termtreatment with potentially toxic drugs would then be required.

The embryonic stem cells of the present invention may be transplanted toa human subject per se, or in a pharmaceutical composition where it ismixed with suitable carriers or excipients.

As used herein a “pharmaceutical composition” refers to a preparation ofone or more of the active ingredients described herein with otherchemical components such as physiologically suitable carriers andexcipients. The purpose of a pharmaceutical composition is to facilitateadministration of a compound to an organism.

Herein the term “active ingredient” refers to the embryonic stem cellsof the present invention accountable for the biological effect.

Hereinafter, the phrases “physiologically acceptable carrier” and“pharmaceutically acceptable carrier” which may be interchangeably usedrefer to a carrier or a diluent that does not cause significantirritation to an organism and does not abrogate the biological activityand properties of the administered compound. An adjuvant is includedunder these phrases.

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of anactive ingredient. Examples, without limitation, of excipients includecalcium carbonate, calcium phosphate, various sugars and types ofstarch, cellulose derivatives, gelatin, vegetable oils and polyethyleneglycols.

Techniques for formulation and administration of drugs may be found in“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

One may administer the pharmaceutical composition in a systemic manner(as detailed hereinabove). Alternatively, one may administer thepharmaceutical composition locally, for example, via injection of thepharmaceutical composition directly into a tissue region of a patient.

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active ingredients intopreparations which, can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the active ingredients of the pharmaceutical compositionmay be formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hank's solution, Ringer's solution, orphysiological salt buffer. For transmucosal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

For oral administration, the pharmaceutical composition can beformulated readily by combining the active compounds withpharmaceutically acceptable carriers well known in the art. Suchcarriers enable the pharmaceutical composition to be formulated astablets, pills, dragees, capsules, liquids, gels, syrups, slurries,suspensions, and the like, for oral ingestion by a patient.Pharmacological preparations for oral use can be made using a solidexcipient, optionally grinding the resulting mixture, and processing themixture of granules, after adding suitable auxiliaries if desired, toobtain tablets or dragee cores. Suitable excipients are, in particular,fillers such as sugars, including lactose, sucrose, mannitol, orsorbitol; cellulose preparations such as, for example, maize starch,wheat starch, rice starch, potato starch, gelatin, gum tragacanth,methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarbomethylcellulose; and/or physiologically acceptable polymers such aspolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acidor a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, titanium dioxide, lacquer solutions and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical compositions which can be used orally, include push-fitcapsules made of gelatin as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules may contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, lubricants such as talc ormagnesium stearate and, optionally, stabilizers. In soft capsules, theactive ingredients may be dissolved or suspended in suitable liquids,such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added. All formulations for oraladministration should be in dosages suitable for the chosen route ofadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by nasal inhalation, the active ingredients for useaccording to the present invention are conveniently delivered in theform of an aerosol spray presentation from a pressurized pack or anebulizer with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichloro-tetrafluoroethane or carbon dioxide. In the case of apressurized aerosol, the dosage unit may be determined by providing avalve to deliver a metered amount. Capsules and cartridges of, e.g.,gelatin for use in a dispenser may be formulated containing a powder mixof the compound and a suitable powder base such as lactose or starch.

The pharmaceutical composition described herein may be formulated forparenteral administration, e.g., by bolus injection or continuousinfusion. Formulations for injection may be presented in unit dosageform, e.g., in ampoules or in multidose containers with optionally, anadded preservative. The compositions may be suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the active preparation in water-soluble form.Additionally, suspensions of the active ingredients may be prepared asappropriate oily or water based injection suspensions. Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acids esters such as ethyl oleate, triglycerides orliposomes. Aqueous injection suspensions may contain substances, whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension may alsocontain suitable stabilizers or agents which increase the solubility ofthe active ingredients to allow for the preparation of highlyconcentrated solutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile, pyrogen-free waterbased solution, before use.

The pharmaceutical composition of the present invention may also beformulated in rectal compositions such as suppositories or retentionenemas, using, e.g., conventional suppository bases such as cocoa butteror other glycerides.

Pharmaceutical compositions suitable for use in context of the presentinvention include compositions wherein the active ingredients arecontained in an amount effective to achieve the intended purpose. Morespecifically, a therapeutically effective amount means an amount ofactive ingredients (nucleic acid construct) effective to prevent,alleviate or ameliorate symptoms of a disorder (e.g., ischemia) orprolong the survival of the subject being treated.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For any preparation used in the methods of the invention, thetherapeutically effective amount or dose can be estimated initially fromin vitro and cell culture assays. For example, a dose can be formulatedin animal models to achieve a desired concentration or titer. Suchinformation can be used to more accurately determine useful doses inhumans.

Toxicity and therapeutic efficacy of the active ingredients describedherein can be determined by standard pharmaceutical procedures in vitro,in cell cultures or experimental animals. The data obtained from thesein vitro and cell culture assays and animal studies can be used informulating a range of dosage for use in human. The dosage may varydepending upon the dosage form employed and the route of administrationutilized. The exact formulation, route of administration and dosage canbe chosen by the individual physician in view of the patient'scondition. (See e.g., Fingl, et al., 1975, in “The Pharmacological Basisof Therapeutics”, Ch. 1 p. 1).

Dosage amount and interval may be adjusted individually to provideplasma or brain levels of the active ingredient are sufficient to induceor suppress the biological effect (minimal effective concentration,MEC). The MEC will vary for each preparation, but can be estimated fromin vitro data. Dosages necessary to achieve the MEC will depend onindividual characteristics and route of administration. Detection assayscan be used to determine plasma concentrations.

Depending on the severity and responsiveness of the condition to betreated, dosing can be of a single or a plurality of administrations,with course of treatment lasting from several days to several weeks oruntil cure is effected or diminution of the disease state is achieved.

The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc.

Compositions of the present invention may, if desired, be presented in apack or dispenser device, such as an FDA approved kit, which may containone or more unit dosage forms containing the active ingredient. The packmay, for example, comprise metal or plastic foil, such as a blisterpack. The pack or dispenser device may be accompanied by instructionsfor administration. The pack or dispenser may also be accommodated by anotice associated with the container in a form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals, which notice is reflective of approval by the agency ofthe form of the compositions or human or veterinary administration. Suchnotice, for example, may be of labeling approved by the U.S. Food andDrug Administration for prescription drugs or of an approved productinsert. Compositions comprising a preparation of the inventionformulated in a compatible pharmaceutical carrier may also be prepared,placed in an appropriate container, and labeled for treatment of anindicated condition, as if further detailed above.

In addition to cell replacement therapy, the embryonic stem cells of thepresent invention can also be utilized to prepare a cDNA library. mRNAis prepared by standard techniques from the embryonic stem cells and isfurther reverse transcribed to form cDNA. The cDNA preparation can besubtracted with nucleotides from embryonic fibroblasts and other cellsof undesired specificity, to produce a subtracted cDNA library bytechniques known in the art.

The embryonic stem cells of the present invention can be used to screenfor factors (such as small molecule drugs, peptides, polynucleotides,and the like) or conditions (such as culture conditions or manipulation)that affect the differentiation of lineage precursor to terminallydifferentiated cells. For example, growth affecting substances, toxinsor potential differentiation factors can be tested by their addition tothe culture medium.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”,

John Wiley & Sons, New York (1988); Watson et al., “Recombinant DNA”,Scientific American Books, New York; Birren et al. (eds) “GenomeAnalysis: A Laboratory Manual Series”, Vols. 1-4, Cold Spring HarborLaboratory Press, New York (1998); methodologies as set forth in U.S.Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057;“Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis, J. E., ed.(1994); “Culture of Animal Cells—A Manual of Basic Technique” byFreshney, Wiley-Liss, N. Y. (1994), Third Edition; “Current Protocols inImmunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al.(eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange,Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods inCellular Immunology”, W.H. Freeman and Co., New York (1980); availableimmunoassays are extensively described in the patent and scientificliterature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153;3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654;3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed.(1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J.,eds. (1985); “Transcription and Translation” Hames, B. D., and HigginsS. J., eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986);“Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide toMolecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol.1-317, Academic Press; “PCR Protocols: A Guide To Methods AndApplications”, Academic Press, San Diego, Calif. (1990); Marshak et al.,“Strategies for Protein Purification and Characterization—A LaboratoryCourse Manual” CSHL Press (1996); all of which are incorporated byreference as if fully set forth herein. Other general references areprovided throughout this document. The procedures therein are believedto be well known in the art and are provided for the convenience of thereader. All the information contained therein is incorporated herein byreference.

Example 1 Generation of Cell Lines Derived from Delayed BlastocystCulture

Materials and Methods

Blastocyst Cultivation:

Discarded zygotes were donated by couples undergoing in vitrofertilization (IVF) treatment at Rambam Medical Center having signedconsent forms approved by the national Helsinki committee. Zygotes werecultured to the blastocyst stage according to IVF laboratory standardprotocol: drops under oil using specialized Cook media (Queensland,Australia) including insemination medium (IM), growth medium (GM) andblastocyst stage embryo medium (BM).

Derivation of Extended Blastocyst Cell (EBC) Lines:

Following zona pellucida digestion by Tyrode's acidic solution (SigmaAldrich, St Louis, Mo., USA) the exposed blastocysts were plated onmitotically inactivated mouse embryonic fibroblasts (MEFs). Eightattached blastocysts were cultured on MEFs as whole embryos for 9-14days post fertilization until a large cyst developed. If needed, due toMEF quality, the embryos were transferred in whole to new MEF-coveredplates using 27 gouge syringe needles, leaving a few of the surroundingfibroblasts behind. Following cyst development, a disc-like structurewas isolated and plated separately on a fresh MEF-covered plate. Cellswith stem cell morphology (small cells with large nucleus) were passagedmechanically. Following a few passages (4-6), when a homogonous culturewas achieved, the cells were passaged routinely every four to six daysusing 1 mg/ml type IV collagenase (Gibco Invitrogen corporationproducts, San Diago, Calif., USA).

Culture Media:

For the derivation and initial passages, cells were grown in a culturemedium consisting of 80% KO-DMEM and supplemented with 20% defined FBS(HyClone, Utah, USA), 1 mM L-glutamine, 0.1 mM β-mercaptoethanol, 1%non-essential amino acid stock (all from Gibco Invitrogen corporationproducts, San Diago, Calif., USA products).

The cells were then cultured using medium consisting of 85% ko-DMEM andsupplemented with 15% ko-serum replacement, 1 mM L-glutamine, 0.1 mMj3-mercaptoethanol, 1% non-essential amino acid stock and 4 ng/ml basicfibroblast growth factor (bFGF) (all products from Gibco Invitrogencorporation products, San Diago, Calif., USA). The cells were frozen inliquid nitrogen using a freezing solution consisting of 10% DMSO (Sigma,St Louis, Mo., USA), 10% FBS (Hyclone, Utah, USA) and 80% KO-DMEM.

Results

Four EBC lines were derived with the use of the extended blastocystculture technique. Of the eight plated embryos, four had a surroundingmonolayer of trophblast or fibroblast cells, and the inner cell mass(ICM) started to grow as a monolayer, creating a typical ES cell colonyfollowing five to ten days (I-5, I-8, I-10, I-11). The other fourembryos continued to develop in whole with a notable cyst and adisc-like area (see FIG. 1E) from which the EBC lines were derived (J2,J3, J6, J7). FIGS. 1A-D are photographs of light microscopy imagesdepicting the generation of EBC J3. FIG. 1E is a light microscopy imageof the EBC J6.

Example 2 Morphological Analysis of EBCs

Materials and Methods

Karyotype Analysis:

Cell division was blocked in mitotic metaphase using colcemid-spindleformation inhibitor (karyoMax colcemid solution, Gibco Invitrogencorporation products, San Diago, Calif., USA). Nuclear membranes werebroken following hypotonic treatment. For chromosome visualization,G-band standard staining (Giemsa, Merck, Darmstadt, Germany) wasperformed. The karyotypes were analyzed and reported according to the“International System for Human Cytogenetic Nomenclature” (ISCN). Atleast 20 cells were examined from each cell line, 10 from each differentsample. The samples were taken from the EBCs following at least 20consecutive passages.

Microscopy:

Light microscopy was performed to examine EBC colonies. Electron andmonolayer microscopy were performed to examine histological sections ofextended blastocyst and embryonic stem cells. The phase-contrast isOlympus CH30, the transmitted electron microscopy in use JEM-100sx(JEOL, Tokyo, Japan).

Results

Karyotype analysis revealed that two of the examined EBC lines compriseda normal 46, XY karyotype and two comprised a normal 46, XX karyotype.None of the 100 cells examined exhibited any karyotype abnormality. Thekaryotype analysis was carried out following at least 20 passages ofcontinuous culture indicating that the cell lines possess a stablekaryotype.

Light microscopy revealed that the colonies formed by the EBCs presenteda similar morphology to that of hESC lines i.e. round colonies withspaces between the cells and relatively large nucleii with distinctnucleoli. However, when cell monolayers were examined under a lightmicroscope, it was noted that while the cells inside the EBC colonieswere organized as a columnar epithelium with somewhat smaller nucleiiand villi throughout the upper side of the colony (FIGS. 2B-C) the ESCcolonies were organized as stratified epithelium, with no villi facingthe upper side (FIG. 2A). Furthermore, electron microscopy indicatedthat zonula occludente junctions, typical for columnar epithelium, couldbe detected between the EBCs and not between the upper rows of hESCs(FIGS. 2D-E). Other reports describe this morphology as the morphologyof early differentiating hES cells [Sathananthan et al. 2002].

Example 3 Differentiational Analysis of EBCs Compared to ESCs

Materials and Methods

Immunostaining:

EBCs and hESCs were fixed with 4% paraformaldehyde, and exposed toprimary antibodies (1:50) overnight at 4° C. Cy3 conjugated antibodies(Chemicon International, Temecula Calif., USA) at a dilution of 1:100were used as secondary antibodies in combination with all primaryantibodies except Brachury where a FITZ conjugated secondary antibodywas used (Santa Cruz). Stage-specific embryonic antigens (SSEA) 1, 3 and4 (Hybridoma bank, Iuwa, USA), tumor recognition antigens (TRA) 1-60 andTRA1-81, (Chemicon International, Temecula, Canada) Brachury and Oct4(Santa Cruz) were used as primary antibodies. For double immunostaining,EBCs and hESCs were fixed with 4% paraformaldehyde, and exposed to Oct4antibody (1:50) for 30 minutes at room temperature. Following washing,the fixed-cells were incubated for 30 minutes at room temperature withBrachury (1:50) antibody. This protocol was repeated with the secondaryantibodies. For each of the reactions a negative control was used inwhich primary antibodies were not used.

Teratoma Formation:

10⁷ cells in 100 μl culture medium were harvested and injected into therear leg muscles of four-week-old male SCID-beige mice. The resultingteratomas were harvested 9 weeks (±2 days) post injection. Each teratomawas weighed, following which a portion of the tumours was collected forRNA isolation and another portion was fixed. The sections were fixed in10% neutral-buffered formalin, dehydrated in graduated alcohol(70%-100%) and embedded in paraffin. For histological examination, 1-5μm sections were deparafinized and stained with hematoxylin/eosin (H&E).

EB Formation:

For the formation of EBs, four to six confluent wells of a six-wellplate (40-60 cm²) of EBCs and hESCs were used. The cells were removedfrom their culture dishes using 1 mg/ml type IV collagenase, furtherbroken into small clumps using 1000 μl Gilson pipette tips, and culturedin suspension in 58 mm Petri dishes (Greiner, Germany). EBs were grownin suspension for five days in medium supplemented with 15% ko-serumreplacement, with similar supplements as the continuous culture mediumwithout the addition of bFGF. After five days in suspension, part of theEBs were trypsinized, further broken up using 1000 μl Gilson pipettetips, and plated on gelatine-coated culture plates. The EB-derived cellswere further cultured in medium supplemented with FBS (Hyclone, Utah,USA) for ten to eleven additional days. Both samples of the EBs culturedin suspension and the plated EB-derived cells were collected for RNAisolation.

RT-PCR Reaction:

Total RNA was isolated from EBCs, five day-old EBs cultured insuspension, plated EB-derived cells and teratoma sections usingTri-Reagent (Sigma Aldrich, St. Louis, Mo., USA), according to themanufacturer's recommended protocol. cDNA was synthesized from 1 μgtotal RNA using MMLV reverse transcriptase RNase H minus (Promega,Madison, Wis., USA). PCR reactions comprised denaturation for 5 minutesin 94° C. followed by repeated cycles of 30 seconds at 94° C., at theannealing temperature (as in Table 2) and for extension at 72° C. PCRprimers and reaction conditions used are as described in Table 2 hereinbelow. PCR products were size fractionated using 2% agarose gelelectrophoresis, following staining with ethidium bromide. RT reactionmixture was used as a negative control, and the β-actin gene (ahouse-keeping gene) was used for normalization.

TABLE 2  Product 5′ primer 3′ primer Mg Annealing Gene size bpSEQ ID NO: SEQ ID NO: mM temp Ref.Undifferentiated markers and receptors Epidermal 300 CAGTCGTCAGCCTGAGGTTGCACTT 1.5  65, Schuldiner et al PNAS growth factor AACATAACATCCGTCCACGCATT AM5 2000 receptor I SEQ ID NO: 1 CCC (EGFR) SEQ ID NO: 2U48727 U48728 Activin 550 ACACGGGAGTGCA TTCATGAGCTG 1.5 65,Schuldiner et al PNAS receptor β2 TCTACTACAACG GGCCTTCCAGA AM5 2000(ACTR) SEQ ID NO: 3 CAC AF060200 SEQ ID NO: 4 Octamer 219 GAGAACAATGAGATTCTGGCGCCG 1.5 55, Abdel-Rahman et al. binding ACCTTCAGGAGA GTTACAGAACCDK4 Hum reprod 1995 protein 4 SEQ ID NO: 5 A (Oct4) SEQ ID NO: 6NM_00270 Nanog 800 ACTAACATGAGTGT TCATCTTCACAC 1.5 61,Daheron et al Stem NG_004095 GGATCC GTCTTCAG AM1 Cells. 2004SEQ ID NO: 7 SEQ ID NO: 8 LIF 560 CAGCATCACTGAAT AGTATGAAACA 1.5  61,Chen et al. Fer Ster X13967 CACAGAGC TCCCCACAGGG AM1 1999. SEQ ID NO: 9SEQ ID NO: 10 LIF-R 459 CAAAAGAGTGTCT CCATGTATTTAC 1.5 61,Chen et al. Fer Ster NM_002310 GTGAG ATTGGC AM1 1999. SEQ ID NO: 11SEQ ID NO: 12 Rex1 306 GCGTACGCAAATT CAGCATCCTAA 1.5 56,Henderson et al. Stem AF450454 AAAGTCCAGA ACAGCTCGCAG 3AM Cells 2002.SEQ ID NO: 13 AAT SEQ ID NO: 14 FGF4 370 CTACAACGCCTACG GTTGCACCAGA 1.552, Henderson et al. Stem NM_002007 AGTCCTACA AAAGTCAGAGT 4AMCells 2002. SEQ ID NO: 15 TG SEQ ID NO: 16 FGF5 550 CACTGATAGGAACCTCCGACTGCTT 1.5 65, Crickard et al NM_004464 CCTAGAGG GAATCTTGG AM5Gynecology Oncology SEQ ID NO: 17 SEQ ID NO: 18 1994 Sox2 448CCCCCGGCGGCAA TCGGCGCCGGG 1.5 60, Henderson et al. Stem Z31560 TAGCAGAGATACAT DK5 Cells 2002. SEQ ID NO: 19 SEQ ID NO: 20 β-Actin 838ATCTGGCACCACAC CGTCATACTCCT 1.5 62 Henderson et al. Stem NM_001101CTTCTACAATGAGC GCTTGCTGATC Cells 2002. TGCG CACATCTGC SEQ ID NO: 21SEQ ID NO: 22 Retinoic 500 AGCAGCAGTTCTG GTGGAGAGTTC 1.5 65,Schuldiner et al PNAS Acid AAGAGATAGTGCC ACTGAACTTGT AM5 2000 ReceptorSEQ ID NO: 23 CCC type alpha SEQ ID NO: 24 (RAR) AH007261 Fibroblast 410AGCATCATAATGG AGTCCGATAGA 1.5 65, Schuldiner et al PNAS GrowthACTCTGTGGTGCC GTTACCCGCCA AM5 2000 Factor SEQ ID NO: 25 AGC ReceptorSEQ ID NO: 26 type I (FGFRI) M34641 Bone 800 TCTGCAGCTAGGTC TATACTGCTCC1.5 65, Schuldiner et al PNAS Morphogenic CTCTCATCAGC ATATCGACCTC AM52000 Protein 4 SEQ ID NO: 27 GGC Receptor SEQ ID NO: 28 type II(BMP4RII) D50516 Hepatocyte 440 AGAAATTCATCAG TTCCTCCGATCG 1.5 65,Schuldiner et al PNAS Growth GCTGTGAAGCGCG CACACATTTGT AM5 2000 FactorSEQ ID NO: 29 CG Receptor SEQ ID NO: 30 (c-Met) AC002080 Nerve 410TGTTCTCCTGCCAG TCTTGAAGGCT 1.5 65, Schuldiner et al PNAS GrowthGACAAGCAGAAC ATGTAGGCCAC AM5 2000 Factor SEQ ID NO: 31 AAGG ReceptorSEQ ID NO: 32 (NGFR) AC006487 Transforming 530 TAGTCACTGACAACACAGTGCTCGC 1.5 65, Schuldiner et al PNAS Growth AACGGTGCAGTCTGAACTCCATG AM5 2000 Factor SEQ ID NO: 33 AGC Receptor SEQ ID NO: 34type II (TGFRII) AH004921 Mesodermal markers β-Globulin 410ACCTGACTCCTGAG TAGCCACACCA 1.5 65, Schuldiner et al PNAS V00499GAGAAGTCTGC GCCACCACTTT AM5 2000 SEQ ID NO: 35 CTG SEQ ID NO: 36Collagen 180 CGATGGCTGCACG CAGGTTGGGAT 1.5 64, B. Schmitt et altype 1 α1 AGTCACAC GGAGGGAGTTT AM7 Differentiation 2003 (cartiladge)SEQ ID NO: 37 AC Z74615 SEQ ID NO: 38 Collagen 128 CCGGGCAGAGGGCCAATGATGGGG 1.5 64, B. Schmitt et al type 2 a1 AATAGCAGGTT AGGCGTGAG AM7Differentiation 2003 (cartiladge) SEQ ID NO: 39 SEQ ID NO: 40 X06268Cartilage 116 GGGTGGCCGCCTG CTTGCCGCAGC 1.5 64, B. Schmitt et aloligomeric GGGGTCTT TGATGGGTCTC AM7 Differentiation 2003 matrixSEQ ID NO: 41 SEQ ID NO: 42 protein (comp) L32137 Cartilage 145GCGTCCGCTACCCC GCGCTCTAAGG 1.5 64, B. Schmitt et al link protein ATCTCTAGCACATTCAGT AM7 Differentiation 2003 U43328 SEQ ID NO: 43 TSEQ ID NO: 44 Aggrecan 146 CCAGTGCACAGAG TCCGAGGGTGC 1.5 64,B. Schmitt et al (cartilage) GGGTTTG CGTGAG AM7 Differentiation 2003X17406 SEQ ID NO: 45 SEQ ID NO: 46 Brachyury 250 TAAGGTGGATCTTCCATCTCATTGGT 2.5 65  P. J. Gokhale et al. Cell NM003181 AGGTAGCGAGCTCCCT AM5 Growth Differ. 2000. SEQ ID NO: 47 SEQ ID NO: 48 Cardiac630 TCTATGAGGGCTAC CCTGACTGGAA 1.5 65  Schuldiner et al PNAS ActinGCTTTG GGTAGATGG AM5 2000 (cACT) SEQ ID NO: 49 SEQ ID NO: 50 NM_005159δ-Globin  430 ACCATGGTGCATCT ACTTGTGAGCC 1.5 65  Schuldiner et al PNAS(δ-Glob) GACTCCTGAGG AAGGCATTAGC AM5 2000 V00505 SEQ ID NO: 51 CACSEQ ID NO: 52 Renin 590 AGTCGTCTTTGACA GGTAGAACCTG 1.5 65 Schuldiner et al PNAS AH007216 CTGGTTCGTCC AGATGTAGGAT AM5 2000SEQ ID NO: 53 GC SEQ ID NO: 54 GATA4 475 AGACATCGCACTG GACGGGTCACT 1  60, Home Made transcription ACTGAGAAC ATCTGTGCAAC DK5 factorSEQ ID NO: 55 SEQ ID NO: 56 D78260 IL-6 628 ATGAACTCCTTCTC GAAGAGCCCTC1.5  54.2  Gutsche et al. Mol NM_00060 CACAAGCGC AGGCTGGACTGHuman Reproduction SEQ ID NO: 57 SEQ ID NO: 58 2003 BMP2 200TCAAGCCAAACAC ACGTCTGAACA 1.5 61, Bunger et al. Calcif NM_001200 AAACAGCATGGCATGA DK3 Tissue Int.2003 SEQ ID NO: 59 SEQ ID NO: 60 BMP4 378GCCGGAGGGCCAA CTGCCTGATCTC 1.5   64.7, Bae et al. D30751 GCGTAGCCCTAAGAGCGGCACCCA DK2 Toxicological Sciences SEQ ID NO: 61 CATC 2003SEQ ID NO: 62 CD44 200 CCAACACCTCCCAC TATACTCGCCCT 1.5 61,Ramos-Nino et al. M59040 TATGAC TCTTGCTG DK3 Cancer Research 2003SEQ ID NO: 63 SEQ ID NO: 64 Endodermal markers Amylase 490GCTGGGCTCAGTAT GACGACAATCT 1.5 65, Schuldiner et al PNAS M24895TCCCCAAATAC CTGACCTGAGT AM5 2000 SEQ ID NO: 65 AGG SEQ ID NO: 66 α1 Anti360 AGACCCTTTGAAGT CCATTGCTGAA 1.5 65, Schuldiner et al PNAS trypsinCAAGGACACCG GACCTTAGTGA AM5 2000 K02212 SEQ ID NO: 67 TGC SEQ ID NO: 68Albumin 450 CCTTTGGCACAATG CAGCAGTCAGC 1.5 65, Schuldiner et al PNASM12533 AAGTGGGTAACC CATTTCACCAT AM5 2000 SEQ ID NO: 69 AGG SEQ ID NO: 70Glucagon 370 CTCAGTGATCCTGA AGTCCCTGGCG 1.5 65, Schuldiner et al PNASX03991 TCAGATGAACG GCAAGATTATC AM5 2000 SEQ ID NO: 71 AAG SEQ ID NO: 72α- 216 GCTGGATTGTCTGCA TCCCCTGAAGA 1.5 60, Home made PhetoproteinGGATGGGGAA AAATTGGTTAA DK5 BC027881 SEQ ID NO: 73 AAT SEQ ID NO: 74 GnRH219 TCAAAAACTCCTAG CTTTCCAGAGC 1.5 55, CTGGCCT TCCTTTCAGGT DK4SEQ ID NO: 75 SEQ ID NO: 76 CG 556 GCAGCTATCTTTCT ACTCTGAGGTG 2.0 60,GGTCACAT ACGTTCTTTTG DK5 SEQ ID NO: 77 SEQ ID NO: 78 Ectodermal markerNeurofilament 400 TGAACACAGACGC CACCTTTATGTG 1.5 65,Schuldiner et al PNAS heavy Chain TATGCGCTCAG AGTGGACACAG AM5 2000(NFH) X15307 SEQ ID NO: 79 AG X15309 SEQ ID NO: 80

Chip DNA Analysis:

Samples of RNA were isolated from confluent cultures of undifferentiatedhESCs and EBCs cultured as described in Example 1 with mitoticallyinactivated MEFs. RNA isolation was conducted as described hereinabovefor RT-PCR analysis. Four commercial gene array membranes were used:human stem cell genes, human TGF_(β)/BMP pathway genes, humanneurotrophin and receptor genes and extracellular molecules genes (allnon-radioactive from GEarray Q series, numbers, HS-018, respectively,SuperArrays Biosciences Inc., Frederick, Md., USA). The analysis wasperformed according to the supplier's instructions. In brief, for RTreaction 200 U Maloney murine leukaemia virus-derived reversetranscriptase was used (Promega, Madisom, Wis., USA). The arraymembranes were hybridized overnight with biotin-labelled probes at 60°C., washed twice at 60° C. for 15 minutes with 2×SSC/1% SDS followedwith 0.1×SSC/1% SDS. The detection stage comprised a 30-minuteincubation of the membranes with alkaline phosphatase-conjugatedstreptavidin followed by a five-minute incubation with CDP-starsubstrate at room temperature. The membranes were exposed to X-ray film.The quantification of the gene expression was carried out using GEarrayanalyzer and Scanalyze software.

Real Time PCR Analysis:

cDNA was synthesized as described above for the RT-PCR reaction. Taq-ManUniversal PCR master Mix and Assay-on-Demand gene expression Probes forOct 4 and β-Actin were used according to the manufacturer instructions(Applied Biosystems, Foster City, Calif.). The real time PCR reactionwas performed using Applied Biosystem 7000 DNA Sequence Detectionsystem, according to the manufacturer guidelines (Applied Biosystems,Foster City, Calif.). The relative expression of Oct 4 was normalizedaccording to the β-Actin expression in cDNA isolated from the samesample, by using the standard curve method described by themanufacturer. To calculate differences in Oct 4 amplification betweenthe various tested cell lines, a relative standard curve method was used(Applied Biosystems, Foster City, Calif.). The data was collected fromthree different samples for each line (I3, I6, J3, J6) and each cDNAsample was assayed at least twice, averaged and graphed with standarddeviations.

Affymetrix Chip Analysis:

RNA was isolated from undifferentiated I3 cells at passage 45, from I6cells at passage 42, from J3 cells at passage 37 and from J6 cells atpassage 49 following culture on MEFs. RNA integrity was assed by gelelectophoresis. Total RNA samples were concentrated at 1 μg/μl, cleanedwith Phenol/chlorophorm/Isoamyl alcohol in Phase lock gel (PLG) tubes(Invitrogen Coorperation, San Diego, Calif., USA). 10 μg of each samplewas used to prepare biotinylated target cDNA and hybridised toAffymetric focus gene chips according to the manufacturer'sinstructions. The microarray was scanned by Affymetric scanner(Affymetrix, Santa Clara, Calif., USA). For bioinformatics the MAS 5.0Affymetrix array analysis software was used. Expression levels ofgreater than 3 fold were considered relevant. The p-value of each spotwas calculated using Anove tests, each SP was centered and normalized.For clustering analysis, the super Paramagnetic Clustering Method wasused. David software was used for the Go-Chart analysis.

Illumina Beadarray Analysis:

RNA was isolated from undifferentiated J3 and from hESC as describedhereinabove for Affymetrix chip analysis. For gene profiling IlluminaBeadarray was used (Sentrix human Expression BeadChip, Illumina, SanDiego, Calif., USA).

2-D Gel Protein Electrophoresis Analysis:

The proteins were dissolved in 150 μl running buffer consisting of: 7Murea, 2M thiourea, 2% w/v CHAPS, 65 mM DTT and 1.25% v/v ampholyte and 3β1 Bromo-Phenol-Blue (BPB). The protein levels were measured using theBradford method. 200 μg of protein were loaded on each strip (BioRad,3-10 NL, Immobiline DryStrips, 11 cm) and rehydrated for one hourwithout voltage and subsequently 12 hours in 50 mV. The isoelectricfocusing was conducted as follows: one hour in 200 V, 500 V, 1000 Vrespectively followed by 30 minutes of increased voltage up to 8000 V,where it remained until reaching 69,145 VH on VH (Volt Hours).

Each strip underwent equilibration in 50 mg DTT dissolved inequilibration buffer containing: 6 M Urea, 30% w/v glycerol and 2% SDSin 0.05 M Tris-HCl buffer (1.5 M Tris-HCl and 0.4% w/v SDS), pH 8.8 10μl BPB, for 15 minutes. The second stage of the equilibration wasconducted in 200 mg Iodoacetamide (IAA) dissolved in equilibrationbuffer and 10 μl BPB, for 15 minutes. The strip were than loaded ontoBio Rad gradient gels (4-20%). A dual color marker was used to evaluatethe molecular weight. Electrophoresis was conducted for one hour at 200V. The resultant gels were stained with SeeBand forte (GeBA) accordingto the manufacturer's instructions. The gels were scanned using Bio RadFluor-S scanner, and analyzed using Bio Rad PDQUEST 7.0.1 software.

Osteoblast Differentiation:

hESC (I4 and I6) and EBC cells (J3 and J6) were cultured on stromal cellmatrix (ATCC no. SR4987) in medium consisting of of 85% MEM alpha, 0.5%Penicillin streptomycin, 0.2% Ribnucleosides & Deoxyribnucleosides(Biological Industries, Bait Haemek, Israel), 15% FBS (HyClonre, Utah,USA), 0.05 mg/ml Ascorbic Acid, 1% β-glycophosphate (Sigma, St. Louis,USA). Following 5 days of co-culture, EBs were formed as described aboveand cultured in suspension in the identical medium for an additional 5-7days. The EBs were then dissociated using trypsin-EDTA solution, andre-plated on stromal cell matrix. Following one week, the cells werestained with Alizarin Red (Fluka, Bochs, Switzerland) to identifycalcium secretion.

Results:

Based on the morphology of the colonies, it was noted that thebackground differentiation of the EBCs is higher than that in theregular hESCs. A consistent background differentiation of more than 15%could be detected in the J2 and J6 cell lines. A single-cell clone ofthe J2 line, J2.1, expressed the same percentage of backgrounddifferentiation as the parental cell line. The J3 differentiation rateswere found to be similar to those of hESCs.

Similar to hESCs, immunostaining of EBCs with embryonic stage-specificmarkers revealed strong staining with SSEA4 and TRA-1-60, no stainingwith SSEA1 and weak staining with SSEA3. Unlike hESC, the EBC linesdemonstrated weak staining with TRA-1-81 (FIGS. 3A-G).

The teratoma model was used to test the EBC pluripotency. The cells wereinjected into the hind limb muscles of four-week-old male SCID-beigemice, similar to the method used to assess the pluripotency of hESCs.During the first set of experiments approximately five million cellswere injected into each mouse with at least four mice per cell line.This showed a reduced success rate of 30% for the EBCs and a 100%success rate for the hESCs. In order to efficiently compare thepluripotency of the different types of cell lines, the number ofinjected cells was doubled to ten million per mouse. In this case, 11 of12 EBC-injected mice resulted in teratomas. The size of the teratomasdiffered between the two types of cell lines as measured by the tumorweight; specifically the average weight of the hESC-resulting teratomaswas 9.4 gr (N=8) and the average weight of the EBC-resulting teratomaswas only 3.5 gr (N=12). In addition to the difference in size, theEBC-resulting teratomas contained a limited number of ectoderm andendoderm representative tissues and had a more distinct appearance ofmesodermal tissues (FIGS. 4A-B). In one case, the J2 resultant teratomadid not contain any ectodermal tissues (one mouse out of threeexamined). While screening several teratoma sections, a tissuerepresentative for all three embryonic germ layers could be found (FIGS.5A-F). In the J6 teratomas, a predominance of cartilage tissues wasobserved (FIG. 6).

The results from the teratoma experiments give the impression that theEBCs have a higher tendency to differentiate into mesencymal tissue thanhESCs. To further confirm this assumption, an in vitro differentiationmodel was used. Cells from both types were cultured in suspension for 5days. The resultant EBs were broken into single cells, plated andfurther cultured for ten additional days as monolayer of differentiatingcells. The expression of several genes was compared by RT-PCR betweenthe different cell lines at different differentiation stages.

Several differences between the EBCs and the ESCs were detected in thenon-differentiated cells. While the EBCs expressed undifferentiatedmarkers such as OCT4, Nanog, Rex 1 and SOX2, they also expressed someearly markers of cartilage differentiation such as COMP, cartilagelinked protein and Aggrecan. The hESCs did not express as manydifferentiation markers at the undifferentiated stage. Anotherdifference was the expression of STAT3 which was highly expressed by theEBCs but was low or non-existent in the hESCs. The results aresummarized in Table 3 below.

TABLE 3 Undifferentiated cells I series J series Gene H9 I6 I3 J3 J6 J2Mesodermal differentiation markers β-Globulin − + + + − − Collagen typeI − + + + + + COMP (Cartilage) −− −− −− +− ++ ++ Collagen type II − −− + + + Cartilage link protein − − − + + + Aggrecan − − − + + + Cardiacactin (cACT) − − + + + + δ-Globulin + − + − − − Renin + − + + + +Brachychyury −− −− −− −+ ++ ++ BMP 4 − − − + + + BMP2 −+ −+ −+ ++ ++ ++Endodermal differentiation markers Amylase + + + − + − α1 Anti trypsin +− + + + + Albumin + + + − − − Glucagon − − − − − − GnRH + + + + + +CGα + + + − − + Ectodermal differentiation marker Neurofilament heavy −− + + + + chain (NFH) Undifferentiated cell markers and receptorsEpidermal GF receptor I ++ ++ ++ +− ++ ++ (EGERI) Activin receptor β2 −− − + + + Retinoic acid receptor + + + + + + type α (RAR) Fibroblast GFreceptor + + + + + + (FGFRI) BMP4RII + − − + + − Hepatocyte GFreceptor + + + + + + C-Met Nerve GF receptor − − − + + + NGFRTransforming GF receptor II + + + + + + (TGF) Oct 4 + + + + + + Rex 1 ++++ ++ ++ ++ ++ Nanong +++ +++ +++ ++− ±++ ++− FGF4 ++− ++− +++ −−− −+−+++ Sox2 −++ +−+ +++ +++ +++ −++

To evaluate possible differences in Oct 4 expression between EBCs andhESCs, a real time PCR analysis was conducted. Although the RT-PCRreaction revealed a clear band for Oct 4 expression for the EBC cells,according to quantitative real-time PCR analysis the levels of Oct 4expression in these cells is significantly lower compared with hESCs(FIG. 7).

To allow a wider comparison between the two cell types, commercial DNAchip analysis was performed using an array of 288 “stem cells genes”(FIGS. 8A-D). When the number of genes with significant expressiondifferences between two different hESC lines and two separate EBC lineswere compared, only minor differences were found of 15 and 9 genes,respectively. When the genes' expression pattern was compared betweenthe average of the EBC lines and hESC lines, 90 genes demonstrateddifferent expression levels. Most of these genes had statisticallyhigher expression in the EBCs—these genes are summarized in Table 4hereinbelow.

TABLE 4 Confirmed I series J series by Gene I6 I3 H9 J2 J6 J3 RT-PCRBone morphogenetic protein +− ND +− ++ ++ ND + 2 (BMP2) NM_001200 Bonemorphogenetic protein − ND − + + ND + 4 (BMP4) NM_001200 CD44 AntigenM59040 − ND − + + ND + Cadherin 3 placental (CDH3) −+ ND −+ ++ ++ NDNM_001793 GATA4 transcription factor − ND − + + ND + D78260 Growthdifferentiation factor − ND − + + ND 3 (GDF3) NM_020634 Interleukin 6(IL6) − ND − + + ND + NM_00060 Integrin beta 5 (ITGB5) − ND − + + NDNM_002213 Leukemia inhibitory factor − ND − + + ND + (LIF) X13967 Nervegrowth factor − ND − + + ND beta polypeptide (NGFB) X52599 NK2 homolog B(NKX2B) − ND − + + ND NM_002509 Hypothetical protein −+ ND −+ ++ ++ ND(FLJ10314) BC039861 Noggin (NOG) NM_005450 − ND − + + ND Notch homolog1translesion − ND − + + ND associated (NOTCH1) AF308602 Patched homolog− ND − + + ND (PTCH) U43148 Sox 13 SRY box 13 (SOX13) − ND − + + NDNM_005686 Sox18 SRY box 18 (Sox 18) − ND − + + ND NM_018419 Sox 2 SRYbox 2 (Sox 2) − ND −+ + + ND −+ BC013923 Transforming growth factor −+ND −+ ++ ++ ND beta receptor III (TGFBR3) NM_003243 THY-1 cell surfaceantigen − ND − ++ ++ ND (THY1) NM_006288 Undifferentiated − ND − + −+ NDembryonic cell transcription factor 1 (UTF1) NM_003577 Wingless-typeMMTV − ND − + + ND integration family 6 (WNT6) NM_006522 Zinc fingerprotein 42 − ND − + + ND (ZFP42) AK056719

Among these genes it was found that BMP2 expression was higher in theundifferentiated EBCs and mesencymal markers such as BMP4, CD44, GATA4(known also as early endodermal marker) were expressed by the twoexamined EBC lines. The expression of these genes was further confirmedby RT-PCR.

Table 5 below summarizes the results of TGF_(β) related gene expressionas analyzed by chip analysis.

TABLE 5 Confirmed I series J series by Gene I6 I3 H9 J2 J6 J3 RT-PCRBone morphogenetic − ND ND ND +/− ND + protein 2 (BMP2) NM_001200 Bonemorphogenetic − ND ND ND ++ ND + protein 4 (BMP4) NM_001200 AntiMullerian −/+ ND ND ND ++ ND hormone (AMH) NM_000479 Anti Mullerian + NDND ND + ND hormone receptor type II (AMHR2) NM_020547 Bone morphogenetic− ND ND ND + ND protein 6 (BMP6) NM_001718 Bone morphogenetic − ND ND ND+/− ND protein receptor type IA (BMPR1A) NM_004329 Bone morphogenetic −ND ND ND +/− ND protein receptor type 2 (BMPR2) Z48923 Cyclin dependent−/+ ND ND ND + ND kinase inhibitor 1A (CDKN1A) L47233 Cerberus 1 (CER1)++ ND ND ND ++ ND NM_005454 Collagen type I ++ ND ND ND ++ ND alpha2(COL1A2) NM_000089 Collagen type III ++ ND ND ND ++ ND alpha1 (COL3A1)NM_000090 Distal-less homeo − ND ND ND ++ ND box 2 (DLX2) NM_004405Lefty A +++ ND ND ND +++ ND (endometrial bleeding associated factor)(EBAF) NM_003240 Bone morphogenetic ++ ND ND ND ++ ND protein 9 = growthdifferentiation factor 2 (BMP9) AF188285 Growth differentiation − ND NDND + ND factor 5 (GDF5, = cartilage derived morphogenetic protein 1)NM_000557 DNA-binding protein −/+ ND ND ND ++ ND inhibitor ID-1 (ID1)D13889 Inhibitor of DNA + ND ND ND +++ ND binding 2, dominant negativehelix-loop- helix protein (ID2) D13891 Inhibitor of DNA −/+ ND ND ND ++ND binding 3, dominant negative helix-loop- helix protein (ID3) X66924Inhibitor of DNA +++ ND ND ND +++ ND binding 4, dominant negativehelix-loop- helix protein (ID4) NM_001546 Inhibin α (INHA) +++ ND ND ND+++ ND NM_002191 Jun B proto- + ND ND ND + ND oncogene (JUNB) X51345Lefty B (LEFTB) +++ ND ND ND +++ ND NM_020997 MAD homolog 2 −/+ ND ND ND++ ND (MADH2) NM_005901 MAD homolog 3 −/+ ND ND ND ++ ND (Smad 3)NM_005902 MAD homolog 6 ++ ND ND ND ++ ND (Smad 6) NM_005585 v-myc avian− ND ND ND ++ ND myelocytomatosis viral oncogene homolog (c-myc) X00364Runt-related ++ ND ND ND ++ ND transcription factor 2 (RUNX2) L40992Plasminogen activator +++ ND ND ND +++ ND inhibitor type I (SERPINE1)M16006 Homo sapiens −/+ ND ND ND + ND teratocarcinoma- derived growthfactor 1 (TDGF1) NM_003212 Transforming growth −/+ ND ND ND +++ NDfactor beta 1 (TGF_(β1)) X02812 TGF_(β) induced factor − ND ND ND ++ ND(TGIF) NM_003244 Tissue inhibitor of − ND ND ND ++ ND metalloproteinase1 (TINP1) (erythroid potentiating activity, collagenase inhibitor)NM_003254 Transforming growth − ND ND ND ++ ND factor beta stimulatedprotein (TSC22) NM_006022

Table 6 summarizes the data accumulated following chip analysis of allExtracellular Matrix related genes.

TABLE 6 Confirmed I series J series by Gene I6 I3 H9 J6 J2 J3 RT-PCRCaveolin 1, + ND ND + ND ND Cavaolae protein 22 KD (CAV1) NM_001753Cadherin 1, Type 1 ++ ND ND ++ ND ND epithelial Cadherin (CDH1) Z13009Carcinoembryonic ++ ND ND ++ ND ND antigen-related cell adhesionmolecule 5 (CEACAM5) NM_004363 Collagen type XVIII ++ ND ND +/− ND ND(Endostatin) AF018081 Collagen type 1 ++ ND ND + ND ND alpha 1 (Col1 A1)NM_000088 Collagen type IV ++ ND ND + ND ND alpha 2 (Col4A2) X05610Cystatin C (amyloid ++ ND ND + ND ND anglopathy and cerebral hemorrhage,CST3) NM_000099 Catenin, Cadherin ++ ND ND + ND ND associated protein,alpha 1 (CTNNA1) NM_001903 Catenin, Cadherin ++ ND ND + ND ND associatedprotein, Beta 1 (CTNNB1) NM_001904 Catenin, Cadherin ++ ND ND ++ ND NDassociated protein, delta 1 (CTNND1) AF062343 Cathepsin B (CTSB) ++ NDND + ND ND L16510 Cathepsin D + ND ND − ND ND lysosomal aspartylprotease (CTSD) M11233 Cathepsin L ++ ND ND + ND ND (CTSL) X12451Fibronectin 1 +++ ND ND +++ ND ND (FN1) X02761 Integrin alpha 5, +++ NDND −/+ ND ND fibronectin receptor (ITGA5) X06256 Integrin alpha 6, −/+ND ND − ND ND laminin receptor (ITGA6) X53586 Integrin alpha V, +++ NDND +++ ND ND vitronectin receptor (ITGAV) NM_002210 Integrin beta 1, +++ND ND +++ ND ND fibronectin receptor (ITGB1) NM_002211 Integrin beta 5,+++ ND ND +++ ND ND (ITGB5) J05633 Laminin B1 chain +++ ND ND +++ ND ND(LAMB1) M61916 Laminin gamma 1 + ND ND − ND ND (LAMC1) Jo3202Hyaluronidases, +++ ND ND + ND ND meningioma expressed antigen 5 (MGEA5)NM_012215 Matrix ++ ND ND − ND ND Metalloproteinase 1 (MMP1) X05231Matrix ++ ND ND − ND ND Metalloproteinase 10 (MMP10) NM_002425Stromelysin-3 + ND ND − ND ND (MMP11) X57766 Matrix + ND ND − ND NDMetalloproteinase 13 (MMP13) X75308 mRNA for +++ ND ND −/+ ND NDmembrane type matrix Metalloproteinase 1 (MMP14) D26512 Matrix +++ ND ND− ND ND Metalloproteinase 15 membrane inserted (MMP15) D86331 Matrix +ND ND − ND ND Metalloproteinase 17 membrane inserted (MMP17) NM_016155Matrix +++ ND ND +++ ND ND Metalloproteinase 2 (MMP2) J03210 Matrix +++ND ND −/+ ND ND Metalloproteinase 24 membrane inserted (MMP24) NM_006690mRNA matrix +++ ND ND +++ ND ND Metalloproteinase 26 (MMP26) AF291664Matrix +++ ND ND − ND ND Metalloproteinase 3 (MMP3) X05232 Neurophilcell + ND ND − ND ND adhesion molecule (NRCAM) NM_005010 Plasminogen +++ND ND +++ ND ND activator inhibitor type 1 (SERPINE1) M16006Osteonectin, +++ ND ND +++ ND ND secreted protein acidic cystein-rich(SPARC) XM_003989 Osteopontin, +++ ND ND +++ ND ND secretedphosphoprotein 1 (SPP1) M83248 Thrombosponin 1 +++ ND ND +++ ND ND(THBS1) NM_003246 Tissue inhibitor of +++ ND ND −/+ ND NDmetalloproteinase 1 (TIMP1) NM_003254 Tissue inhibitor of +++ ND ND ++ND ND metalloproteinase 2 (TIMP2) NM_003255 Tissue inhibitor of ++ ND ND−/+ ND ND metalloproteinase 3 (TIMP3) NM_000362

Table 7 summarizes the data accumulated following chip analysis of allLIF pathway related genes.

TABLE 7 Confirmed I series J series by Gene I6 I3 H9 J6 J2 J3 RT-PCRBCL2-associated X + ND ND + ND ND protein (Bax) L22474 Cerebellin 1 + NDND + ND ND precursor (CBLN1) NM_004352 Corticotropin releasing + ND ND +ND ND hormone (CRH) NM_000756 Corticotropin releasing −/+ ND ND + ND NDhormone binding protein (CRHBP) NM_001882 Corticotropin releasing ++ NDND ++ ND ND hormone receptor 1 (CRHR1) NM_004382 Corticotropin releasing++ ND ND +++ ND ND hormone receptor 2 (CRHR2) NM_001883 Chemokinereceptor 4 + ND ND + ND ND (CXCR4) NM_003467 Prothrombin kringle-1 + NDND + ND ND (F2) J00307 Basic fibroblast −/+ ND ND −/+ ND ND growthfactor (FGF2) NM_002006 Fibroblast growth + ND ND + ND ND factor 9(FGF9) XM_007105 Fibroblast growth + ND ND + ND ND + factor receptor 1(FGFR1) M34185 Fusion derived from + ND ND ++ ND ND t(12; 16) malignantliposarcoma (FUS) NM_004960 Heat shock 27 KD + ND ND + ND ND protein(HSPB1) Z23090 Interleukin 6 (IL6) − ND ND −/+ ND ND M14584 Interleukin6 signal + ND ND + ND ND transducer, gp130 oncostatin M receptor (gp130)NM_002184 v-jun avian sarcoma ++ ND ND +++ ND ND virus 17 oncogenehomolog (v-jun) NM_002228 Leukemia inhibitory + ND ND + ND ND + factorreceptor mRNA (LIFR) NM_002310 Leukemia inhibitory − ND ND − ND ND Wasfactor (LIF) X13967 demonstrated with RT- PCR for J6 Neurotrophin + NDND + ND ND receptor-interacting MAGE homologue (MAGED1) NM_006986 Nervegrowth factor + ND ND + ND ND beta polypeptide (NGFB) X52599Neuropeptide Y + ND ND + ND ND receptor Y6 (NPY6R) NM_006173Suc1-associated + ND ND + ND ND neurotrophic factor target 2 (FGFRsignaling adaptor) (SNT-2) NM_006653 Signal transducer −/+ ND ND ++ NDND and activator of transcription 3 (STAT3) NM_003150 Tumor protein P53−/+ ND ND + ND ND (p53) M14694 Urocortin (UCN) ++ ND ND +++ ND NDNM_003353

The difference between EBC and hESC gene expression could be the resultof either the higher background differentiation of the EBCs compared tothat of the hESCs, as mentioned earlier, or a significant differencebetween the two cell lines stemming from the different embryonicdevelopmental stage. To explore the two possibilities, double stainingfor OCT4 and early mesodermal marker—Brachyury [Herrmann et al. 1990;Wilkinson et al. 1990; Herrmann 1991] was performed. It washypothesized, that if the difference was due to backgrounddifferentiation, then in some colonies only several EBCs would bepositive for Brachyury and that these cells would be negative for OCT4.A double expression of the two markers would indicate that the secondpossibility is more likely to be true. First, the expression ofBrachyury was measured in undifferentiated cells by RT-PCR. Only line J6expressed this gene in three separate samples, whereas J2 and J3expressed it inconsistently. The hESCs did not express it in any of theexamined nine samples from three different lines. No double staining wasfound in undifferentiated colonies of the hESC line I3. It was foundthat almost all the cells were positive for OCT4 and few were positivefor Brachyury (FIGS. 9A-I). However, when undifferentiated colonies ofthe line J6 were examined, most of the cells were double stained (FIGS.10A-I).

The gene profiles of undifferentiated hES cells from lines I3 and I6,and undifferentiated EB cells from lines J3 and J6 were compared usingAffimetrix focused DNA chips (Affymetrix U133A GeneChip DNA microarray).Clustering analysis of the four lines showed a distinct expression(FIGS. 11A-B). Lines I3 and I6 exhibited a significantly higher numberof “present” calls (According to the MASS method of analysis, “present”call genes are those whose 11 representative expression spots averaged ascore higher than 30) than J3 and J6. No differences were found betweenthe number of “present” calls of I3 compared with I6, or J3 comparedwith J6, although some individual gene expression signals were found tobe different. This finding is consistent with other reportsdemonstrating that embryonic stem cells express a higher number of genescompared with other progenitors (such as hematopoietic stem cells orskin stem cells); these genes “switch off” during the differentiationprocess [Mashiach et al, 2005, FASEB 19:147-9]. The signal intensitiesof lines J3 and J6, however, were found to be higher than the signalintensities produced by the hESC lines. A list of 238 genes in lines J3and J6 was found to be significantly different (either increased ordecreased three fold) than the average expression of I3 and I6 asexamined by t-test. The gene list also includes the early mesodermalmarker Brachyury, further corroborating both the RT-PCR results and thedouble staining with Oct 4. Go-chart analysis of these 238 genesrevealed that the majority of these genes are related to cell growth,cell maintenance, metabolism of proteins and nucleic acid and signaltransduction pathways (FIG. 12). The signal transduction genes include 4genes from the WNT signaling pathway, which were found to be decreasedin line J3 and J6.

To allow an even wider comparison between the two cell types, illuminebead array analysis was performed using an array of 24,364 genes. Theresults are summarized in Table 8 below (J3—cells of the presentinvention; hES-human embryonic stem cells; Shh—Chip control; sd—standarddeviation; av—average). It was found that 2498 of them showed a 5 foldchange in expression level. Of these, only 696 genes (27%) wereexpressed at a higher level (greater than 5 fold) in the stem cells ofthe present invention. The average expression level of those 696 geneswas much lower than the average expression level of those genes whereexpression was higher in ESCs than the stem cells of the presentinvention. The same result appears when expression changes by at least15 fold is examined; 1354 genes are expressed in the hESC while only 411(30%) in the DBC cells.

Lengthy table referenced here US09206392-20151208-T00001 Please refer tothe end of the specification for access instructions.

Using two-dimensional gel electrophoresis, extracted proteins fromundifferentiated cells, and 5-day-old EBs from I6, I4, J3 and J6 lineswere compared. Clear differences were found between the treatments(FIGS. 13A-B). More differences were found between the EBs derived fromEBCs compared with corresponding undifferentiated EBCs than the EBsderived from hESCs compared with corresponding undifferentiated ESCs.These differences include both protein appearance and post translationaldifferences such as phosphorylation.

Directed differentiation into osteoblasts was also tested. Theseexperiments revealed higher staining of calcium in EBs derived from EBCscompared with EBs derived from hESCs. Examples are illustrated in FIG.14.

CONCLUSIONS

Like hESCs, EBCs sustain a normal karyotype, express most of the typicalES cell surface markers, are capable of undifferentiated prolongedproliferation and demonstrate the ability to differentiate into thethree embryonic germ layers progeny.

On the other hand, EBCs hold a significantly different geneticsignature, including expressing early mesodermal markers at theundifferentiated stage, demonstrating a tendency to differentiate intomesodermal tissues, having an altered expression profile, having a lowertendency to form teratomas, having an altered single cell and colonymorphology, and having a reduced pluripotency in comparison with hESCs.

It appears that EBCs represent a distinct cell-population related toearly stage of embryonic development, as compared to hESCs.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents and patentapplications mentioned in this specification are herein incorporated intheir entirety by reference into the specification, to the same extentas if each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated herein byreference. In addition, citation or identification of any reference inthis application shall not be construed as an admission that suchreference is available as prior art to the present invention.

LENGTHY TABLES The patent contains a lengthy table section. A copy ofthe table is available in electronic form from the USPTO web site(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US09206392B2). Anelectronic copy of the table will also be available from the USPTO uponrequest and payment of the fee set forth in 37 CFR 1.19(b)(3).

What is claimed is:
 1. A method of generating a lineage specific cellcomprising culturing an isolated extended blastocyst cell line underconditions: (i) that allow spontaneous differentiation; (ii) that allowformation of teratoma; or (iii) that direct differentiation into cellsof the mesoderm lineage, the ectoderm lineage or the endoderm lineage,wherein said isolated extended blastocyst cell line exhibits for atleast 20 passages an undifferentiated proliferative state, the abilityto differentiate to derivatives of each of an endoderm, mesoderm, andectoderm tissue and a double staining expression of brachyury andOctamer binding transcription factor 4 (OCT-4), but not of SSEA-1,thereby generating the lineage specific cell.
 2. The method of claim 1,wherein said isolated extended blastocyst cell line further expresses atleast one cartilage marker.
 3. The method of claim 2, wherein said atleast one cartilage marker is selected from the group consisting ofCOMP, aggrecan and collagen type II.
 4. The method of claim 1, whereinsaid isolated extended blastocyst cell line maintains a stable normalkaryotype for at least one year.
 5. The method of claim 1, wherein saidisolated extended blastocyst cell line expresses SSEA4 and TRA-1-60markers.
 6. The method of claim 1, wherein said isolated extendedblastocyst cell line expresses less TRA-1-81 marker than an embryonicstem cell of the same primate species not expressing brachyury usingidentical assay conditions.
 7. The method of claim 1, wherein saidisolated extended blastocyst cell line is capable of colony organizationof columnar epithelium with villi throughout the upper side of saidcolony.
 8. The method of claim 1, wherein said isolated extendedblastocyst cell line exhibits an OCT4 protein level lower than the OCT4protein level in an embryonic stem cell of the same primate species notexpressing brachyury using identical assay conditions.
 9. The method ofclaim 1, wherein said isolated extended blastocyst cell line expressesmore mesodermal differentiating markers than an embryonic stem cell ofan identical primate not expressing brachyury using identical assayconditions.
 10. The method of claim 1, wherein said isolated extendedblastocyst cell line being genetically modified.
 11. The method of claim1, wherein said inducing is effected in vitro.
 12. The method of claim1, wherein said inducing is effected ex-vivo.
 13. The method of claim 1,wherein said lineage-specific cell comprises cardiomyocytes.
 14. Themethod of claim 1, wherein said lineage-specific cell comprises neuralor glial lineages.
 15. The method of claim 1, wherein saidlineage-specific cell comprises hematopoietic cells.
 16. The method ofclaim 1, wherein said lineage-specific cell comprises insulin-secretingbeta cells.
 17. The method of claim 1, wherein said differentiation isdirected by genetic modification.
 18. The method of claim 1, whereinsaid differentiation comprises formation of embryoid bodies.
 19. Themethod of claim 1, wherein said primate is a human.